EP1080184A1 - Method for activating natural killer (nk) cells - Google Patents

Abstract

The invention concerns a method for activating natural killer cells which consists in contacting NK cells with dendritic cells in vitro, ex vivo or in vivo. The invention also concerns cell compositions, comprising in particular activated NK cells, NK cells-dendritic cells cocultures or dendritic cells, and their use for stimulating cytolitic activity of NK cells or natural immunity in vivo. The invention further concerns a factor stimulating the NK cells present in the membrane of dendritic cells, and media and factor(s) triggering dendritic cells and their use, whether separate or combined, stimulating NK activity, particularly in vivo. The invention can be used to control the activity of NK cells in vitro, ex vivo or in vivo, particularly in pathological conditions.

Description

 ACTIVATION METHODS FOR NATURAL KILLER CELLS (NK)

The present invention relates to the field of biology and immunology It relates more particularly to new methods for preparing activated natural killer cells and the means of implementing these new methods, in particular new populations of dendritic cells and products derivatives It also relates to the use of activated cells obtained by the methods of the invention, in the fields of immunology, immunotherapy or more generally of medical biotechnology. Natural killer cells (“NK cells”) are a population of lymphocytes which represent a very early line of defense against viruses and tumor cells. NK cells can be characterized by the presence of markers CD56 and CD16, and by the absence of the CD3 marker. NK cells have in particular been implicated in non-specific antitumor immunity of antigens, for the prevention of the establishment of primitive or metastatic tumors in the immunocompetent or immunosuppressed host. In particular, the role of NK cells in immunity surveillance (tumor) primitive or metastasis) has been suggested in mice bearing tumors and treated with IL-2 and / or IL-12 / IL-15, with or without LAK cells (“killer cells activated by lymphokines”), adherent NK cells ( "A-NK") or nonadherent ("NA-NK") - obtained ex vivo by stimulating NK cells with large doses of IL-2. NK cells in particular seem to have a key role against tumor cells or MHC class I negative variants

Because of their non-specific cytotoxic antigen properties and their efficiency, NK cells therefore constitute a population of effective cells particularly interesting for the development of immunoadoptive approaches for the treatment of cancers or infectious diseases. Adoptive anti-tumor immunotherapy have been described in the prior art. Thus, in certain indications such as patients with intensified malignant lymphomas, the administration of Λ-NK with low doses of IL-2 has given promising results in the adjuvant situation. NK cells have also been used for the experimental treatment of various tumor types and some clinical studies have been initiated (Kuppen et al., Int. J Cancer. 56 (1994) 574; Lister et al., Clin. Cancer Res. 1 (1995) 607; Rosenberg et al., N Engl J Med. 316 (1987) 889). In addition, these cells can also be used in vitro for the non-specific lysis of cells not expressing MHC class-1 molecules, and more generally of any cell sensitive to NK cells.

However, adoptive therapy using NK cells (for the treatment of human or human tumors or other pathologies such as infectious diseases) or any other in vitro or in vivo use of these cells involves the expansion and activation ex vivo of NK cells In this respect, current techniques for activating NK cells are all based on the use of cytokines, generally at high doses poorly tolerated by the host. Indeed, the available data seem to indicate that NK cells ex vivo do not survive and cannot be activated without feeder support or cytokines

Thus, current methods of activating NK cells in vitro involve the culture of these cells in the presence of different cytokines (such as, for example, IL-1, IL-2, IL-12, IL-15, IFNα, IFNγ , IL-6, IL-4, IL-18 under certain circumstances), alone or in combination, activation which can be considerably increased by adhesion or costimulation factors such as ICAM, LFA or CD70 Similarly, in vivo , the efficacy of NK cells in tumor immunity cannot be dissociated from the co-administration of cytokines such as IL-2 / IL-15 or IL-12, IL-18, and IL-10 IFNs also have an activating role in association with IL-2 The activation methodologies described in the prior art are therefore all dependent on the use of cytokines. However, these methods have certain drawbacks, linked in particular to the costs of preparing cytokines, toxic character of many cytokines, which cannot be used in in vivo applications, or else the non-specific nature of many cytokines, the use of which in vivo risks being accompanied by numerous undesirable effects In addition, the "natural killmg" function is often impaired in patients with tumors, the ability to harvest these cells for ex vivo activation can be significantly reduced

There is therefore a real need for new methods of activating NK cells. The present application provides a solution to this problem by proposing new original approaches for the activation of NK cells. The present application demonstrates in particular, for the first time , the possibility of activating NK cells at rest with another population of cells The present application also describes, for the first time, a method of activating NK cells not dependent on the presence of cytokines, and therefore making it possible to overcome to the disadvantages described in the prior art The present invention therefore detects new methods for preparing activated natural killer cells and the means for implementing these new methods

A first aspect of the invention therefore resides in a method of activating NK cells comprising bringing NK cells into contact with dendritic cells. As indicated below, contacting can be carried out in vitro, ex vivo or in vivo It can include either the coculture of NK cells and dendritic cells in vitro, or the incubation of NK cells in vitro or in vivo with a preparation derived from dendritic cells, in particular membrane vesicles (exosomes) or a factor for stimulating NK cells from dendritic cells, either the passive in vivo transfer of dendritic cells, or also the in vivo administration of one or more growth factors of dendritic cells

Another aspect of the invention relates to the use of dendritic cells or of a preparation derived from dendritic cells, for the activation of natural killer cells in vitro, ex vivo or in vivo

Another aspect of the invention relates to the use of dendritic cells or of a preparation derived from dendritic cells, for the preparation of a composition intended for activating natural killer cells in vivo. Another aspect of the invention also relates to a coculture of dendritic cells and NK cells

An additional aspect of the invention relates to a composition comprising a factor for stimulating NK cells originating from dendritic cells Another aspect of the invention relates to a method of triggering

("tπggeπng") of dendntic cells to improve (or provoke) their capacity to stimulate NK cells, as well as a medium or a trigger factor for dendntic cells and any composition comprising it The invention also relates to a new population of cells dendntiques, called "triggered" (or "tπggered Dendritic cells"), as well as any composition containing them and their uses

Other aspects of the invention are in particular a subpopulation of NK cells activated by the method of the invention and the use of these cells or of NK / DC co-cultures to stimulate in vivo or in vitro cytotoxic activity against target cells sensitive to NK cells According to another aspect, the invention also relates to methods allowing the major increase in the cytolytic activity and secretπce of IFNg of NK cells at rest

The invention also relates to new therapeutic approaches, in particular for the treatment of infectious, tumor, autoimmune, congenital or transplant-linked pathologies, for example The methods of the invention involve in particular the passive transfer (i) of activated NK cells with dendntic cells ex vivo, or (n) dendntic cells (in particular triggered dendntic cells) or a preparation derived from dendntic cells, to directly activate NK cells in situ, or (m) of the two populations cells co-incubated ex vivo, or (iv) the administration of the "trigger" factor of the dendntic cells to trigger the dendntic cells in vivo in such a way that they become capable of effectively activating the NK cells, factor administered alone or in combination with chemokines or cytokines or dendntic cell growth factors, for example, or administration of NK cell stimulating factor or dendntic cell growth factor, alone or in combination

As indicated above, a first subject of the invention therefore relates to a process for the activation of NK cells by means of dendntic cells. This process notably comprises bringing NK cells into contact with dendntic cells or a preparation derived from cells. The present invention derives in particular from the applicant's demonstration of the ability of dendntic cells to activate NK cells at rest

The results presented in the present application demonstrate in particular that the resting NK cells, co-cultivated in the presence of dendntic cells, survive and are very strongly activated for their lytic and IFNγ production capacity. In addition, the activated cells thus obtained lyse sensitive NK targets but not sensitive LAK targets, which differentiates this activation phenomenon from the classic LAK, IL-2 dependent phenomenon The present application also demonstrates that allogeneic as well as autologous dendntic cells are capable of activating cells in vitro NK, and NK cell activation mechanisms in the presence of dendntiques cells or a preparation derived from dendntiques cells, do not involve I ^'1L-12 or IL-2, or IL 15 nor IFNα On the other hand, the results presented also show the involvement of an interaction between NK cells and the membranes of dendntic cells in the process. sus activation, demonstrating the existence of a factor membrane of dendntic cells involved in this activation The results also show that membrane vesicles produced by dendntic cells are also capable of activating NK cells, which indicates that the stimulating factor of NK cells expressed by dendntic cells is present in said vesicles

The relevance of this activation of NK cells by dendntic cells has also been demonstrated in vivo, in a MHC class I negative tumor model. In the mice carrying this tumor, the only expansion of dendntic cells allows the elimination of the tumor. In an NK-dependent manner In addition, the passive adoptive transfer of dendntic cells to the immunosuppπme animal deficient in T and B cells, carrying the tumor, also allows a significant slowing down of the tumor growth.

These results therefore demonstrate that dendntic cells or preparations derived from dendntic cells, have the capacity to induce activation of NK cells, in vitro, ex vivo or in vivo, that this activation makes it possible to stimulate cell lysis in vitro. NK-sensitive and in vivo natural immunity of a host organism, and can thus lead to the in vivo elimination of tumors, cells infected, or involved in other pathological processes (autoimmune diseases, transplant rejection, reaction graft versus host, etc.) The results obtained in the present invention are all the more surprising since, until now, few or no studies have suggested that NK cells could be activatable, in vitro, by a other cell type, except for transfectants coding for IL-2 and or IL-12, IL-5 and CD70 On the contrary, certain studies seemed rather to suggest a role inhibiting macrophages on the activation IL-2 dep of NK cells, via PGE2

The present invention constitutes, to our knowledge, the first demonstration of an activation of NK cells, not dependent on cytokines, and using another cell population or a preparation or a factor derived therefrom

The term "activation" of NK cells within the meaning of the invention more particularly designates the increase in the production of IFNγ and / or in the cytotoxic activity of NK cells. These two parameters can be easily measured according to the techniques known in the art. skilled in the art and illustrated in the examples. Generally, activation according to the invention is not accompanied by a significant increase in the proliferation of NK cells, all populations combined, but could induce the proliferation of a sub-population of these On the other hand, this activation is accompanied by a significant increase in the survival of NK cells in vitro m More particularly, the activation of NK cells within the meaning of the invention is independent of the use of conventional cytokines The term “activated” NK cells means, within the meaning of the invention, NK cells exhibiting at least one of the properties mentioned above

The method of activating NK cells according to the present invention can be implemented in vitro, ex vivo or directly in vivo

Activation of NK in the presence of dendntic cells

In a first particular embodiment, the method of the invention comprises the activation of NK cells in vitro or ex vivo by coculture of NK cells with mature or immature dendntic cells, autologous or allogenic, preferably triggered according to this first mode implementing the method of the invention, the cells

NK are therefore co-cultivated with dendntic cells In this mode of implementation, the dendntic cells used can be either autologous (that is to say come from the same subject as NK cells), or allogeneic (it is ie from another subject of the same species) Indeed, the results presented in the examples show that the activation of NK cells is not significantly affected by the allogenic character of dendntic cells This allows in a particularly advantageous to use, for the activation of NK cells, “universal” banks of dendntic cells. Such banks can be constituted, as described below, for example by modification of the dendntic cells so as to make them immortal In this regard , different dendntic cell lines can be used for the implementation of the present invention, preferably established from dendnt cells immature human ics

Prior to their use, it is also possible to subject the dendntic cells to pre-treatments in order to improve their properties or to make them compatible with a pharmacological use. Thus, the dendntic cells can be subjected to irradiation, prior to their use for the activation of NK cells. Such irradiation makes it possible in particular to eliminate any carcinogenic risk associated with certain populations of dendntic cells such as immortalized dendntic cells. Pretreatment by irradiation can be particularly desirable when dendntic cells or co-cultures are used in vivo Another pretreatment of dendntic cells can consist of an incubation in the presence of stimulating factors of dendntic cells (cytokines, chemokmes, heat shock protein for example), so as to improve their activity of stimulating NK cells or so as to trigger the production of dexosomes

Preparation and use of triggered dendntic cells

A particularly advantageous treatment of dendntic cells comprises the treatment of these cells in the presence of a triggering medium. The present invention indeed describes the production and the characterization of a new population of dendntic cells having greatly improved NK stimulation capacities. dendntic cells, called "triggered", their preparation and their uses constitute another object of the present application

The term "triggering" designates, within the meaning of the present invention, the presence of a signal, different from the modulation of their stage of differentiation, which induces in dendntic cells a strong capacity for stimulating NK cells. The "triggering medium "can therefore include any substance, generally biological, capable of supplying the dendntic cells with a signal inducing a high capacity to stimulate the NK cells. The dendntic cells thus treated are also designated in the present application by" triggered "(or" tDC ") dendntic cells. for "tπggered dendritic cells")

A particular object of the invention therefore relates to a method of treating dendntic cells comprising bringing dendntic cells into contact with a triggering medium, making it possible to improve (or provoke) their capacity for stimulating NK cells

Another object of the invention also resides in a medium for triggering dendntic cells, that is to say a medium making it possible to improve (or to provoke) the capacity of dendntic cells to stimulate NK cells.

A triggering medium suitable for the present invention can comprise, for example, cells capable of providing the appropriate signal to dendntic cells, a preparation derived from such cells, a factor derived from such cells or any substance, preferably biological, capable of triggering dendntic cells The triggering medium advantageously further comprises growth factors and / or cytokines, in particular GM- CSF and / or interleukιne-4, as well as, where appropriate, constituents of a mammalian cell culture medium (serum, vitamins, amino acids, etc.)

Among the cells capable of being used for the triggering of dendntic cells, one can more particularly mention the extracellular matrix, and in particular the stromal, endothelial or fibroblastic cells These cells, more particularly when they are in the division phase, make it possible to trigger dendntic cells It is the same for certain tumor cells, such as for example mastocytoma cells A particular embodiment of the invention comprises the use of fibroblasts for triggering The Applicant has indeed shown that fibroblasts allow cells Dendntiques to stimulate with much more efficiency the cells NK In this respect, they can be immortalized fibroblasts, transformed lines, primary cultures, activated or not, prepared in advance or extemporaneously, etc. Preferably, the cells used for DC-trigger cell coculture are dividing or likely to divide Among the fibroblast lines which can be used, mention may be made, for example, of the NIH 3T3, L-929, MRC5 or TIB80 lines, for example

For the implementation of this method, the fibroblasts (or other cells) used can be autologous, allogenic or xenogenic with respect to dendntic cells. Indeed, surprisingly, the results presented in the examples show that dendntic cells human can be triggered by fibroblasts of a different species, in particular by muπns fibroblasts In this mode of implementation (in vitro coculture), the triggering (treatment) can be carried out in a DC / cell ratio varying from 0.1 to approximately 100, preferably from 0.5 to 10, in particular from 1 to 5 It is understood that this ratio can be adapted by a person skilled in the art. In addition, in this embodiment, irradiated cells are preferably used ( for example between 2000 and 8000 rad)

As a triggering medium, it is also possible to use, instead of the intact cells, a preparation or a factor derived therefrom. It is therefore possible to use, for example, a supernatant, a lysate, an acellular preparation, a factor. isolated and / or purified, etc. The results presented in the examples show in particular that a fibroblast culture supernatant makes it possible to trigger the dendntic cells, that is to say to make them capable of activating NK cells at rest very effectively (especially faster) In this embodiment, use is made, for example, of approximately 1 to 20 ml of a cell supernatant (for example of fibroblasts) for 10 ⁶ dendntic cells, in a final volume of approximately 30 ml It is therefore possible to use a diluted supernatant 1, 5 to 30 times, preferably 2 to 5 times These conditions can of course be adapted by humans of the profession as a function of the cell populations used Furthermore, the activity described above for the culture supernatant demonstrates the existence of a soluble factor responsible for or sufficient to trigger, secreted by cells, in particular fibroblasts A other triggering medium therefore comprises for example a concentrate / filtrate of supernatant, more particularly a soluble factor mentioned above, in an isolated and / or purified and / or recombinant form. Thus, as triggering medium, it is possible to use the soluble factor described above or a recombinant cell expressing this factor II is also possible to use any other substance, in particular biological, per putting on trigger dendntic cells for activation of NK cells

Generally, the triggering is carried out by incubation of the dendntic cells in the presence of the triggering medium, for a period which can range from 15 to 72 hours, more usually from 20 to 48 hours approximately. The "triggered" state of the dendntic cells does not cause modification of their immunological phenotype Thus, immature dendntic cells remain immature after triggering (expression of HLA-DR, CD40, CD80, CD86, CD83, CD1a) The "triggered" stage can be highlighted in different ways, and generally in testing the capacity of the cells to activate the NK cells in vitro, as described in the examples, or by assaying the triggering factor soluble in the culture supernatant

Furthermore, in the context of the activation of NK cells, the triggering of dendntic cells can be carried out prior to incubation in the presence of NK cells, or concomitantly with it

As indicated above, the present invention also relates to a population of so-called triggered dendntic cells. In this regard, an object of the invention therefore also resides in a composition comprising triggered dendntic cells, in particular human, mature or immature, this is that is to say dendntic cells having an increased capacity for stimulating NK cells, in particular by a factor of at least 2 compared to non-triggered dendntic cells The dendntic cells triggered according to the invention are more particularly defined in that they activate the NK cells at rest and in that they are capable of being obtained by treatment of mature or immature dendntic cells in the presence of a medium or a trigger factor as defined above, preferably comprising a culture of cells of the extracellular matrix or a supernatant of such cells

CD-NK co-cultures

For the implementation of the activation method of the NKs of the invention, the dendntic cells used can be mature dendntic cells (in particular in a muπn system) or preferentially immature cells (in the human and muπn systems) As shown in the examples, dendntic cells have the property of activating NK cells whatever their stage of maturity, in particular after triggering as described above. In order for activation to be more effective, it is advantageous to respect certain parameters such as the NK cell ratio on dendntiques cells and / or the time of comcubation Thus, experiments pa ^r the applicant have shown that the best method for activating performance in vitro or ex vivo are obtained when the initial report NK cells on dendntic cells is between 0.01 and 10, preferably between 0.05 and 5 It is understood that man of the same tier is able to adapt this ratio according to the cell populations used, taking into account the smothering effect of NK cells which can be observed when the quantity of dendntic cells is too large, and the low level of activation which can be observed when the number of dendntic cells is too low. Particularly preferred conditions are those in which the initial ratio of NK cells to dendntic cells is between 0.1 and 1, more preferably from 0.1 to 0.5 approximately.

Regarding the coculture time, it can also be adapted by the skilled person according to the cell populations used and in particular the stage of maturation and triggering of dendntic cells Generally, the optimal activation of NK cells is observed after coculture for a period of between 18 and 48 hours approximately Preferably, when the dendntic cells are triggered as described above, the activation of the NK cells at rest is obtained after a coculture period of less than 20 hours The coculture periods indicated above allow in particular to obtain the best combination between the proportion of activated NK cells and the proportion of viable cells. It should be noted in this regard that, during the period of activation by dendntic cells, no significant overall proliferation of NK cells are not observed (factor 2 approximately) which does not exclude the isolated and specific proliferation of an NK subpopulation In addition, in a particularly unexpected manner, it appears that dendntic cells also exert a positive effect on the survival of NK cells in culture Therefore, the process of the invention makes it possible to obtain activated NK cells, without compulsory recourse to cytokines, and with improved yields Similarly, the NK cells in return increase the survival of mature DCs

The activation of NK cells and the triggering of dendntic cells in vitro can be carried out in any device suitable for cell culture, preferably under sterile conditions. It can in particular be plates, flask culture dishes, flasks, bags, etc. The co-culture is moreover carried out in any medium suitable for the culture of dendntic cells and of NK II cells can more generally be culture media available commercially for the culture of mammalian cells, such as for example the medium RPMI, DMEM medium, IMDM medium or GBEA medium (AIMV, X-VIVO), etc.

According to a first particular variant of the method of the invention, the in vitro or ex vivo activation of NK cells is carried out by coculture of mature dendntic cells with NK cells

In a typical experiment, dendntic cells derived from bone marrow by treatment with GM-CSF + IL-4, matured in LPS, or cells of an established lineage of dendntic cells, in the mature state, are resuspended in their medium culture at 1 million / ml They are then placed in culture in plates or any other appropriate device. Fresh NK cells at rest (autologous or allogenic), obtained after adhesion step, are resuspended in an appropriate medium (RPMI medium supplemented with example) at a concentration of 1 million / ml They are then added to the plate containing the dendntic cells, so that the initial NK CD ratio is approximately 0.1 to 0.3. The coculture is collected at 18-36 approximately hours The activated character of the NK cells is checked by measuring the production of IFNγ in the supernatant and measuring the cytotoxicity against the target cells. The NK cells are also counted (for example le in trypan blue) and analyzed (for example in flow cytometry) for the expression of markers characteristics (such as NK1 1 D x 5, or asιalo-GM1 in mice) and to assess cell mortality

According to another particular variant of the process of the invention, the activation in vitro or ex vivo of NK cells is carried out by coculture of immature dendntic cells with NK cells

In a typical experiment, the cells are treated in an identical manner to that described above, but they are not incubated in the middle of maturation, so as to keep the dendntic cells at an immature stage. In this embodiment, the coculture is advantageously maintained for at least 36-72 hours to allow optimal activation of the NK cells. The activated NK cells can then be analyzed and controlled as described above.

In a preferred embodiment of the method of the invention, the in vitro or ex vivo activation of NK cells is carried out by coculture of dendntic cells triggered with NK cells. In this embodiment, a coculture of less than 20 hours is sufficient to allow optimal activation of the NK cells. The activated NK cells can then be analyzed and controlled as described above. More preferably, these are immature dendritic cells preincubated in the presence of a triggering medium. Even more preferably, it is acts of preincubated immature dendntic cells in the presence of fibroblasts or a fibroblast supernatant or a protein factor produced by fibroblasts

When the NK cells have been thus activated, it is possible either to separate the NK cells from the dendntic cells, or to collect directly the coculture NK cells dendntic cells In this regard, the invention also relates to a composition comprising NK cells and dendntic cells, in particular a coculture NK cells dendntic cells As indicated above, they are advantageously activated NK cells In addition, they can be mature or immature dendntic cells, preferably triggered Finally, in these compositions according to the invention, the cell populations are preferably autologous, that is to say from the same organism. These compositions advantageously consist of isolated cell populations, that is to say that each of the two cell populations is composed less than 10%, preferably at least 30%, especially at least 50% of the corresponding cell type (NK or dendriti that) Furthermore, the preferred compositions according to the invention generally comprise at least 10%, preferably from 20 to 60%, even more preferably from 30 to 60% of NK cells, and at least 40%, preferably from 40 to 80% of dendntic cells The invention also relates to any composition comprising NK cells activated as described in the present application The compositions of the invention can be packaged in any suitable device such as bags, vials, ampoules, syringes, vials, etc., and can be stored (cold) or used extemporaneously, As described below Advantageously, these compositions comprise from 10 ⁴ to 10 ⁹ NK cells, preferably from 10 ⁶ to 10 ° approximately (in particular for administration in humans) or also from 10 ⁵ - 10 ⁶ (in particular for administration in mice)

Activation of NK in the Presence of a Preparation Derived from CD In another embodiment, the method of the invention comprises the activation of NK cells in vitro, ex vivo or in vivo by bringing NK cells into contact with a derivative preparation of dendntic cells The preparation derived from dendntic cells can be any preparation or membrane fraction of dendntic cells, a cell lysate of dendntic cells, membrane vesicles of dendntic cells, or the stimulating factor derived from dendntic cells, in isolated, enriched or purified Indeed, as illustrated in the present application, the activation of NK cells can be obtained not only in the presence of intact dendntic cells, but also of membrane preparations of these, in particular of membrane vesicles (eg, dexosomes), or also a protein stimulating factor. According to a first variant, the p The process of the invention comprises the activation of NK cells in vitro, ex vivo or in vivo by bringing NK cells into contact with membrane vesicles produced by dendntic cells. In this regard, it has been demonstrated that dendntic cells produce membrane vesicles, with a diameter generally between 50 and 100 nm, designated dexosomes (FR9709007, FR9801437) The present invention now shows that these vesicles are also endowed with the activity of stimulating NK cells In particular, the present application shows that dexosomes produced from human or human dendntic cells are capable of activating human NK cells In addition, results show that this activation is observed even with dexosomes produced by non-triggered dendntic cells

Preferably, for the implementation of this variant, use is made of dexosomes produced from immature dendntic cells, preferably autologous or allogenic. For in vitro or ex vivo applications it is preferable to use the dexosomes in a concentration range varying from 10 to 100 μg of exosomal proteins per one million NK cells The amount of exosomal proteins can be easily determined by a person skilled in the art, for example by the Bradford test (Annal Biochem 72 (1976) 248) More preferably on uses the dexosomes at a concentration greater than or equal to 15 μg / 10 ⁶ NK cells, even more preferably greater than or equal to 20 μg / 10 ⁶ NK cells II it should be noted that these concentrations can be adapted by a person skilled in the art, and can be transposed to in vivo uses In particular, for in vivo use, doses of exosomes greater than 50 or 100 can be used μg / ιnjectιon The dexosomes can be prepared according to the techniques described in applications FR9709007 and FR9801437, for example, which are illustrated in the examples Briefly, the dendntic cells are cultured, preferably in the immature stage, preferably in a medium which promotes production dexosomes The dexosomes are then isolated by methods such as centπfugation (in particular by differential ultracentπfugation at 70,000 g), or any other technique known to those skilled in the art. The dexosomes are thus isolated, aliquoted and preserved or used extemporaneously for stimulation. NK cells

According to another variant, the method of the invention comprises the activation of NK cells in vitro, ex vivo or in vivo by bringing NK cells into contact with a stimulating factor originating from dendntic cells, in particular triggered dendntic cells

The results presented in the present application indeed illustrate the specific nature of the activation of NK cells by dendntic cells, and therefore indicate the involvement of one or more factors produced or expressed by dendntic cells in achieving this effect. In this regard, the present application also shows, in a "transwell" experiment, that intercellular contact between NK cells and dendritic cells or a membrane preparation derived therefrom seems necessary for activation. These results therefore illustrate clearly the existence of a stimulating factor for NK cells expressed (at the surface) by dendntic cells and dexosomes, responsible for or at least necessary for this activation of NK cells This (co-) stιmulatιon (membrane) factor, or any acelluiaire preparation comprising it, or any derivative or recombinant forms of this factor as well as the corresponding nucleic acids, can therefore also be used in vitro or in vivo to activate NK cells, in particular for applications in antitumor or antiviral immunization This factor can moreover be blocked by the use of a competitor, of specific antibodies or of antisense, in certain situations such as graft versus host disease or transplant rejection

Another subject of the present invention also relates to a composition comprising a factor for stimulating NK cells derived from CD, in particular a membrane factor involved in the activation of NK cells by dendntic cells. The term "involved" means that this factor is necessary or at least participates in the activation of NK cells by dendntic cells This composition is for example composed of an acelluiaire extract of dendntic cells comprising said factor, or of membrane vesicles or of any isolated or purified form of this factor Le The term "derivative" indicates that this factor, which is essentially protein in nature, can in particular be purified by various isolation methods well known to those skilled in the art, such as cell lysis, followed by various stages of centπfugation (differential centπfugations, ultracentπfugations, etc.), and / or chromatography, electrophoresis, production of neutralizing antibodies and their use for isolation by immunoaffinity, etc. Each of these techniques, alone or in combination (s), can be used to isolate the stimulating factor involved in activation, by following the different stages purification by an NK cell activation test as described in the present application Another approach for the identification of this factor lies in the use of a DNA library of dendntic cells, and in research of clones endowed with activity or capable of complementing mutants of dendntic cells deficient for this activity In this perspective, differential DNA libraries (by subtraction between a triggered dendritic cell and an unlit dendritic cell) can be established

More generally, the present invention now describes a process for the preparation of factors capable of stimulating NK, involving intermembrane contact between NK cells and a test composition. More particularly, the invention relates to a method of identification and / or factor preparation stimulation of NK cells, comprising contacting a biological material comprising a membrane fraction and a population of NK cells, demonstrating activation of NK cells, and isolation of the activation factor present in biological material More preferably, the biological material comprising a membrane fraction can be a dendritic cell, a subcellular preparation of a dendritic cell (in particular a membrane vesicle), or a cell transformed by a nucleic acid encoding a polypeptide product. act from mammalian cells (for example from COS cells) transformed by a DNA library of human origin, in particular from a cDNA library of dendntic cells The clones inducing a stimulation of the activity of NK are selected, and the insert they contain is isolated, purified and characterized This method therefore makes it possible to clone any nucleic acid encoding a fact r of stimulation of NK cells The nucleic acid obtained can be modified, introduced into an expression vector, and used in a process for the production of a protein-stimulating factor

The invention therefore also relates to a method of identification and / or preparation of a factor for stimulating NK cells, comprising contacting a biological material comprising a membrane fraction and a population of NK cells, the evidence of activation of NK cells, and isolation of the activation factor present in biological material

A particular composition of the present invention therefore comprises a factor for stimulating NK cells derived from CD, of an essentially protein nature, capable of being obtained from membranes of mature dendntic cells or from membrane vesicles produced by immature dendntic cells A composition more particular comprises a factor which is essentially protein in nature, capable of being obtained from exosomes produced by immature human dendntic cells, and capable of stimulating the secretion of gamma interferon by NK cells at rest

Furthermore, the term "derivative" also indicates that the compositions of the invention can comprise any variant or recombinant form of the stimulation factor identified above, in particular expressed from a recombinant cell in culture, such as a yeast or mammalian cell

In this regard, the compositions of the invention can also contain any nucleic acid coding for the membrane stimulating factor of cells. dendntiques (in particular triggered) as described above This nucleic acid can be obtained by any technique known to those skilled in the art, and in particular from the DNA library of triggered dendntic cells Finally, the compositions according to invention may also contain any other factor for co-stimulating NK cells, and in particular any lymphokine or cytokine capable, in combination with the stimulating factor described above, of activating NK cells

According to a particular embodiment, the invention therefore comprises a method of activating NK cells in vitro, ex vivo or m vivo by bringing NK cells into contact with a stimulating factor derived from dendntic cells The invention relates therefore, in addition, the use of the stimulation factor as described above for the preparation of a composition intended for increasing the cytolytic activity of NK cells or the production of IFNv and / or TNFα in vivo

The invention also relates to the use of the stimulating factor as described above for the preparation of a composition intended for increasing the natural immunity of an organism

The invention also relates to the use of the stimulation factor as described above for increasing the cytolytic activity of NK cells or the production of IFNv and / or TNFα in vitro or ex vivo

The invention also relates to a method for negatively controlling the activation of NK cells in vitro or in vivo comprising bringing NK cells into contact with a compound capable of interfering (ie, at least partially inhibiting) with the interaction between NK cells and dendntic cells Such a compound can comprise, for example, a soluble form (soluble receptor) or any other fragment of the stimulating factor (in particular the extracellular domain, in particular the binding site), an analog, a antagonist, competitor, antibody or fragment of antibody, antisense, etc. Such a compound can be identified and / or characterized in a screenmg test based on the stimulation factor described above or in any direct or indirect functional test NK activation

In this regard, the invention also relates to a method of identifying and / or characterizing a compound capable of inhibiting the activation of NK cells, comprising the incubation of a test compound or of a composition comprising a or several test compounds, with resting NK cells and dendritic cells, preferably triggered, or dexosomes, or an NK stimulation factor as described above, the measurement of the activation of NK cells, and the selection of compounds / compositions capable of inhibiting (ie, reducing) the activation of NK cells, compared to a control experiment carried out in the absence of test compound / composition The invention therefore also relates to the use of compounds inhibiting this activation of NK by dendntic cells, as a drug or pharmaceutical composition for external use (ex vivo) or internal use (in vivo)

In this regard, the invention also relates to any compound capable of interfering with the interaction between dendntic cells and NK cells, and therefore at least partially inhibiting contact between a dendritic cell (or a dexosome) and a cell. NK This compound can be, as described above, a neutralizing antibody, a competitive ligand, an analogue, fragment or derivative of the stimulating factor, any chemical molecule, etc. The invention also relates to the use of such a compound for controlling the activation of NK cells in vivo or in vitro, in particular in applications such as the prevention of transplant rejection and of GVH In addition, the invention also relates to the use of dendntic cells of "tolerogenic" phenotype, such as that GC-DC, described in particular by Grouard et al (Nature 384, 364-367, 1996) or a product derived from tolerogenic dendntic cells to inhibit the activation of NK cells

Activation of NK by increasing CD in vivo

In another embodiment, the method of the invention comprises the activation of NK cells in vivo, by increasing the levels of dendntic cells in vivo. This increase in vivo makes it possible in fact to exercise in situ activation of NK cells and therefore to strengthen the natural immunity of an organism, in particular against tumor or infected cells

The in vivo increase in dendntic cell levels can be achieved by in vivo administration of dendntic cells, possibly triggered (passive transfer) or also by in vivo administration of one or more growth and / or triggering factors of dendntic cells, optionally in combination This administration is carried out for example by injection, preferably by subcutaneous or systemic injection The injection is preferably a local or regional injection, in particular an injection at the site or close to the site to be treated, in particular close to a tumor The results presented in the examples demonstrate in particular that the administration by subcutaneous or intravenous injection of dendntic cells in vivo, immature or mature, allogenic or autologous, at the site of the tumor, makes it possible to reduce the growth of MHC tumors. class I negative The injections are generally carried out on the basis of doses of cells which can range from 10 ⁴ to 10 ⁹ dendntic cells, preferably between 10 ⁵ and 10 ⁷ inclusive In addition, the injection protocol can be adapted by the skilled in the art depending on the situation (preventive, curative, isolated tumors, metastases, significant or localized infection, etc.) Thus, it is possible to passively transfer dendntic cells by repeated administrations, for example 1 to 2 administrations per week For many months

The increase of dendntic cells in vivo can also be carried out by injection of growth factor of dendntic cells in vivo. Such factors are for example the compound Flt3L (designated in the following compound "FL"), described by Lyman SD et al. , (Blood 83, 2795-2801, 1994, Clonmg of the human homologue of the muπne Flt3L a growth factor for early hematopoietic progenitor cells) and Maraskovsky E et al, (J Exp Med 184, 1953-1962, 1996) or GM -CSF, for example As shown in the examples, stimulation of natural immunity (and therefore of NK cell activity) in vivo is obtained after daily injection of FI13L The present results further show that this injection produces an increase of the absolute number of NK cells in situ, and induces antitumor effects, which are dependent on the lymphoid dendntic cells and on the interaction B7 / CD28 and IFNγ in vivo As indicated below, the compound FL may furthermore s be advantageously associated with the triggering factor of dendntic cells

This embodiment therefore constitutes another particularly effective approach for increasing the cytolytic activity of NK cells in vivo. This approach may advantageously be associated with the co-administration of a growth factor for NK cells in vivo. The invention therefore has The subject of the invention is also the use of dendntic cells for the preparation of a composition intended to activate NK cells in vivo. The invention also relates to the use of dendntic cells for the preparation of a composition intended to activate the cytolytic activity of NK m cells in vivo and the production of IFNγ and or TNFα by activated NK cells As indicated above, the dendntic cells used for this purpose are mature or immature cells, autologous or allogenic, in particular triggered. In addition, it can also be dendntic cells sensitized to one or more antigens

The invention also relates to the use of a dendntic cell growth factor for the preparation of a composition intended to activate NK cells in vivo as well as for the preparation of a composition intended to activate the cytolytic activity of NK cells in vivo The growth factor is preferably the compound FL The growth factor can advantageously be associated with the triggering factor of dendntic cells in vivo The the invention also relates to the use of a protein factor, or more generally a biological factor, for triggering dendntic cells for direct in vivo activation of NK cells, optionally in combination with a growth factor for dendntic cells and or chemokine (s) or cytokines

In a particular embodiment of the method of the invention, the increase in the levels of dendntic cells in vivo is carried out either by administration in vivo, under the conditions described above, of dendntic cells triggered in vitro as described above ( passive transfer) or also by in vivo administration of one or more growth factors of dendntic cells and one or more trigger factors of dendntic cells This embodiment makes it possible to improve the efficiency of activation of NK cells in vivo, insofar as the cells or compounds administered make it possible to obtain in vivo significant levels of triggered dendntic cells

In this regard, the invention also relates to a composition comprising at least one trigger factor for dendntic cells and one growth factor for dendntic cells, as described above, for their simultaneous, separate or spaced-apart use over time. More particularly, such a composition comprises the compound FL and a preparation derived from fibroblasts comprising a soluble triggering factor. More particularly, it comprises the recombinant soluble factor. The invention also relates to the use of such a composition for the preparation of a composition intended to activate NK cells in vivo as well as for the preparation of a composition intended to activate the cytolytic activity of NK cells in vivo For their use, these compounds can be packaged in any suitable medium (saline solutions, buffers , etc), preferably isotonic, and in any device known to those skilled in the art tier (vial, vial, tube, syringe, pouch, etc.)

Preparation of NK cells at rest

For the implementation of the present invention, the NK cells can be obtained by different techniques known to a person skilled in the art. More particularly, these cells can be obtained by different methods. isolation and enrichment from mononuclear cells of peripheral blood (lymphoprep, leukapheresis, etc.) Thus, these cells can be prepared by Percoll density gradients (Timonen et al, J Immunol Methods 51 (1982) 269), by negative depletion methods (Zarling et al, J Immunol 127 (1981) 2575) or by sorting steps by FACS (Lanier et al, J Immunol 131 (1983) 1789) These cells can also be isolated by column immunoadsorption using an avidme-biotin system (Handgretmger et al, J Clin Lab Anal 8 (1994) 443) or by immunoselection using microbeads grafted with antibodies (Geiselhart et al, Nat Immun 15 (1996-97) 227) It is also possible to use combinations of these different techniques, possibly combined with adhesion methods on plastic

These different techniques make it possible to obtain cell populations highly enriched in resting NK cells, preferably comprising more than 70% of resting NK cells. More preferably, the populations of NK cells used for implementing the invention generally comprise more than 30% of NK cells, advantageously more than 50% The purity of the cell populations can be improved if necessary by using specific antibodies such as anti-CD56 antibodies and or anti-CD16 antibodies and / or anti-CD3 antibodies (depletion)

The NK cells can be stored in culture medium in frozen form for later use. Advantageously, the NK cells are prepared extemporaneously, that is to say that they are used for activation after their obtaining.

Preparation of dendntic cells

The dendntic cells used in the context of the present invention can be prepared according to different techniques. These cells can be immature or mature cells, autologous or allogenic, "naive" or sensitized to one or more particular antigens, preferably triggered. dendntic cells used can be cultures of cells enriched in dendntic cells, or even cell cultures essentially comprising dendntic cells Advantageously, these are obviously human dendntic cells

The preparation of dendntic cells has been well documented in the literature. Thus, it is known that these cells can be obtained from cells. hematopoietic strains or from monocyte precursors, or directly isolated in differentiated form (Review by Hart, Blood 90 (1997) 3245)

Obtaining dendntic cells from stem cells is illustrated for example by Inaba et al (J Exp Med 176 (1992) 1693) in mice, and by Caux et al (Nature 360 (1992) 258) or Berπhard et al (Cancer Res 55 (1995) 1099) in humans This work shows in particular that dendntic cells can be produced by bone marrow culture in the presence of Granocyte-Macrophage Colon Stimulation Factor (GM-CSF) or, more precisely , from hematopoietic stem cells (CD34 +) by culture in the presence of a combination of cytokines (GM-CSF + TNFα + IL-3 and IL-4 or else in CD40L)

Obtaining dendntic cells from monocyte precursors is illustrated for example by Romani et al (J Exp Med 180 (1994) 83), Sallusto et al (J Exp Med 179 (1994) 1109), Inaba et al (J Exp Med 175 (1992) 1157) or Jansen et al (J Exp Med 170 (1989) 577) These methodologies are essentially based on the removal of mononuclear cells from the blood and culture in the presence of different combinations of cytokines A particular method consists in treating the monocyte precursors of the blood in the presence of combinations of cytokines such as lnterleukιne-4 + GM-CSF or lnterleukιne-13 + GM-CSF for example This technique is also illustrated by Mayordomo et al, 1995 (muπn) Furthermore, it is also possible to treat the monocyte precursors with pharmacological agents of cell differentiation, such as calcium channel activators or CD40L directly

Another approach for obtaining dendntic cells consists in isolating, from biological samples, dendntic cells already differentiated. This approach has been described for example by Hsu et al (Nature Médiane 2 (1996) 52) The methodology described by this team essentially consists of collecting peripheral blood samples and treating them with different gradients and centπfugations so as to extract the dendntic cells

The preferred methodology in the context of the present invention is based on the production of dendntic cells from monocyte or bone marrow precursors These methodologies are illustrated in the examples More particularly, it is preferred to use in the context of the present invention dendntic cells obtained by breastfeeding monocyte precursors (contained in the blood or marrow) in the presence of a GM-CSF + IL-4 or GM-CSF + IL-13 combination As indicated above, for the implementation of the present invention, it is possible to use a population of dendntic cells comprising immature and / or mature dendritic cells. Advantageously, a population of dendntic cells composed mainly (ie, at least 60%, preferably 70%) of immature dendntic cells, preferably triggered The immature state of dendntic cells corresponds to an early stage of their development, at which they exhibit strong endocytic activity and express low levels of class I molecules and II of the MHC and of lymphocytic co-stimulation molecules on their surface Furthermore, it is also possible to use, within the framework of the present invention, dendntic cell lines II can be immortalized dendntic cell lines produced by example by introduction of an oncogene into dendntic cells II can act as muπn example of such a line, of the following lines described in the prior art line D1 (Wiπzler et al, J Exp Med 185, 317-328, 1997), line XS (A Takashima et al, J Immunol 1995, Vol 154 5128-5135), tsDC line (Volkmann et al., Eur J Immunol 26 2565-72, 1996) The advantage of using dendntic cell lines lies in the constitution of "universal" cell banks, which can be used industrially to activate populations of allogeneic NK cells from different subjects In this embodiment, the line is preferably maintained in 30% of triggering medium or with an optimal concentration of triggering factor

When the dendntic cells are prepared, they can be maintained in culture, further purified, stored or used directly in the implementation of the present invention (activation in vitro, ex vivo or in vivo of NK cells, production of extracts acelluiaire, dexosomes, obtaining the membrane stimulation factor, etc.) In addition, prior to their use for the activation of NK cells, the dendntic cells thus prepared can be sensitized to an antigen or to a group of antigens The presence of antigenic patterns on the surface of dendntic cells could indeed improve (by cross-pπming) or inhibit (in a KIR mode) their immunogenic activity (acquired immunity), especially in the case of in vivo uses

In this perspective, different techniques can be used to sensitize dendntic cells to antigens. These techniques include in particular - bringing the dendntic cells into contact with aigenogenic peptides ("pulsing peptide") This approach consists in incubating the dendntic cells, for a variable time (generally from 30 minutes to 5 hours approximately) with one or more antigenic peptides, ie that is to say with a peptide derived from an antigen, as it could result from the treatment of said antigen by a cell presenting the antigen This type of approach has been described for example for antigenic peptides of the HIV virus, Infiuenza or HPV or for peptides derived from the antigens Muc-1, Mail, Her2 / Neu for example (Macatonia et al, J Exp Med 169 (1989) 1255, Takahashi et al, Int Immunol 5 (1993) 849 , Porgador and Gilboa, J Exp Med 182 (1995) 255, Ossevoort et al, J Immunother 18 (1995) 86, Mayordomo et al, supra, Mehta-Damani et al, J Immunol (1994) 996) It is also possible d incubate dendntic cells with an acid peptide eluate of a tumor cell according to the methodology described by Zitvogel et al (1996, cited above)

- bringing the dendntic cells into contact with one or more antigens ("antigen pulsing") This approach consists in incubating the dendntic cells not with one or more antigenic peptides, but with the intact antigen (s) The advantage of this technique lies in the fact that the antigen will be transformed into antigenic peptides by the natural mechanisms of the dendritic cell, so that the antigenic peptides resulting and presented by the dendritic cell should provide better immunogenicity This approach has been illustrated for example by Inaba and al (J Exp Med 172 (1990) 631) or by Hsu et al, (Nature Medicine 2 (1996) 52)

- bringing the dendntic cells into contact with one or more antigenic protein complexes This approach is similar to the previous one but can make it possible to improve the efficiency of transformation and / or presentation of the antigen. In particular, the antigen can be used in soluble form or complexed with targeting elements, making it possible in particular to target membrane receptors such as mannose receptors or immunoglobulin receptors (RFc) (immune complexes) It is also possible to make the antigen particulate so as to improve its penetration or even its phagocytosis by the cells

- bringing the dendntic cells into contact with cells (hybπdome-like fusion) or membranes, lysates or sonicates of cells expressing antigens or antigenic peptides This technique is based on the direct transfer of antigens or antigenic peptides by fusion of cells or membranes This approach has been illustrated for example by the fusion between dendntic cells and tumor cell membranes (Zou et al, Cancer Immunol Immunother 15 (1992) 1, and Gilboa, supra)

- bringing the dendntic cells into contact with membrane vesicles containing antigens or antigenic peptides (in particular exosomes of tumor cells as described above) This approach for sensitizing dendntic cells using exosomes, as demonstrated in the present invention, is particularly advantageous insofar as it does not require the knowledge of the particular antigens and where the charged antigenic peptides are in a native conformation This technology is illustrated in the examples

- bringing the dendntic cells into contact with liposomes containing antigens or antigenic peptides (Nair et al, J Exp Med 175 (1992) 609)

- bringing the dendntic cells into contact with RNAs coding for antigens or antigenic peptides, (see Boczkowsky et al, 1996, cited above)

- bringing the dendntic cells into contact with DNAs coding for antigens or antigenic peptides or nucleic acid sequences coupled with protein antigens (possibly incorporated into vectors of the plasmid, viral or chemical type) Thus, a mode of sensitization dendntic cells consists, for example, of infecting dendntic cells with a virus against which protection is sought. This has been described for example for the influenza virus (Bhardwaj et al, J Clin Invest 94 (1994) 797, Macatonia et al Another approach consists in delivering, by means of a virus or other nucleic acid transfer vectors, a DNA coding for the antigen (s) or antigenic peptides of interest. Such an approach has been illustrated for example. by Arthur et al (Cancer Geπe Therapy, 1995) or by Alijagie et al (Eur J Immunol. 25 (1995) 3100) Certain viruses such as adenoviruses, AAVs or retrov irus seems to be able to be used for this purpose, to deliver a nucleic acid in a dendritic cell

Applications

The invention also relates to the use of the methods, cells and compositions described above, in particular in the fields of immunology, immunotherapy or medical biotechnology As indicated above, these There are many uses, both in vitro and in vivo, to control the activity of NK cells. Such applications are in particular the treatment of various pathologies such as cancers or infectious diseases, in particular viral or to other pathogens, autoimmune diseases, pathologies linked to transplantation (transplant rejection, GVHD), congenital diseases (deficits for receptors to interferon or to interleukin-12 for example), etc. The processes, cells and compositions of the invention are in particular usable for retarding the growth or even suppressing tumors (in particular tumors weakly expressing molecules of class I of the MHC) or other pathological cells. For this type of applications, as indicated above, the compositions cells (activated NK cells, NK / DC cocultures or dendntic cells) can be administered loco-regionally, preferably by sub-cu tanee or systemic The doses of cells are indicated above as well as in the experimental part which follows The invention is also usable m vitro for the treatment of cellular preparations, in particular for the destruction of cells sensitive to NK cells The invention can also be used in combination or as an adjuvant of immunizations based on the development of an antigen-specific cytotoxic T lymphocyte activity The invention further relates to the use of dendntic cells or of a dendntic cell membrane factor to increase the survival of NK lymphocyte populations, m vitro, ex vivo or in vivo, as well as to increase, if necessary, the proliferation of NK lymphocyte subpopulations The invention further relates to the use of NK cells or of a cell membrane factor NK to increase the survival of mature dendntic cells, in vitro, ex vivo or in vivo,

Other advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

LEGEND OF FIGURES

FIGURE 1 Immature dendntic cells stimulate NK activity in vitro Autologous immature dendritic cells derived from mouse marrow

BALB / c, that is to say cultivated in GM-CSF and inter-leukene-4, were incubated 24h in 30% L-929 fibroblast medium or else co-cultivated with irradiated L-929 After 24h, they were counted , resuspended in conventional medium (GM-CSF + IL-4) and co-incubated for a period of 40 to 72 hours at a concentration of 1 million cells per ml in round bottom wells, in 96-well plates, with splenic cells (of which 10-30% are NK) at rest, originating from the spleen of the syngeneic souces SCID BALB / c at the same concentration Cytotoxicity against the YAC and P815 target cells as well as the release of interferon γ by the NK cells are determined as described in the Materials and Methods

FIGURE 2 mature dendntic cells directly stimulate NK activity in vitro

(a) Dendntic cells of splenic origin (derived from the spleen of C57BL / 6 animals) mature very strongly stimulate the cytolytic activity of the cells

NK Dendntic cells incubated in the presence of L-929 supernatant (30%) then, for 24 hours in a medium containing TNFα, were co-cultured with non-adherent mononuclear cells of the spleen from B6-Rag mice - / - or SCID mice in a 1 1 ratio for 40 to 48 hours The viable lymphocytes were tested against YAC-1 cells in a chromium 51 release test for 4 hours The results are expressed by the percentage of specific lysis at different effective cell / target cell ratios These experiments were performed 5 times with similar results

(b) Dendntic cells of mature splenic origin stimulate the production of interferon γ by NK cells. Supernatants of dendntic cells or syngeπic or allogenic NK cells cultivated separately or together at different concentration ratios were collected at time 48. at 72 hours and tests for the presence of interferon Y muπn by ELISA Note in this figure that the NK CD ratios correspond in fact to the splenic cell ratios of animals depleted en in T / B / Mac (containing 10-30% NK pure) CD No trace of γ interferon was detected in the supernatant of NK cells or of dendntic cells cultured separately as controls

FIGURE J Activation involves contact of an NK cell with a dendritic cell Study of the specific lysis of target cells (YAC-1) induced by NK cells activated in vitro by a line of dendntic cells, either by coculture (filled triangles) or in a "Transwells" system in which the two cell populations are physically separated by a porous membrane (empty triangles) and are at 1 mm distance from each other The controls are represented by the NK and dendntic cells grown separately

FIGURE 4 The administration of FL in Nude B6 mice carrying an AK7 tumor induces a significant suppression of tumor growth

10 μg of FL were administered daily for 20 days to B6-nude mice carrying a tumor established on day 20 of AK7 mesothelioma The tumor growth was checked twice a week for 60 days The average sizes of the tumors for groups of 5 mice are shown in the figure with the standard deviation This experiment was repeated four times with suppression of similar tumor growth Similar results were obtained in Rag2 B6 mice - / -

FIGURE 5 The anti-tumor effects induced by FL are dependent on NK cells (a) The compound FL has no effect in B6-Belgian mice 10 μg of FL were administered daily for 20 days in B6-Belgian mice bearing an AK7 tumor established by day 20 The tumor growth was checked twice a week for 50 days The average size of the tumors for groups of 5 mice is represented in the figure with the standard deviation This experiment was repeated twice with identical results

(b) The co-administration of neutralizing monoclonal antibody antι-NK1 1 at the same time as FL inhibits the anti-tumor effect of FL 300 μg per mouse of anti-NK1 1 antibodies were co-administered daily to mice immunocompetent B6 carrying a tumor established for 20 days at the same time as the compound FL The groups of mice treated with a PBS phosphate buffer exhibited similar tumor growth kinetics in the beige mice and in the mice Nude (*) represents significantly more tumors large in groups of mice depleted by NK1 1 in comparison with animals treated with FL only. This experiment was reproduced 2 times with identical data

FIGURE 6 LF therapy is accompanied by increased NK activity in spleens of B6 mice

The splenocytes of mice without tumor or carrying an AK7 tumor were prepared after 20 days of treatment in phosphate buffer (PBS) or FL, and used as effectπces cells in a chromium release test using YAC-1 cells as target cells The results are expressed by the percentage of specific lysis at different target effector ratios Each group includes 3 mice

FIGURE 7 Role of lymphoid dendntic cells, of the B7 / CD28 interaction and of cytokines associated with Th1 differentiation in the anti-tumor effect dependent on NK cells induced by FL

(a) Depletion experiments in vivo using anti-CD8α or anti-CD4 antibodies in B6-nude mice B6 nude mice bearing tumors were treated with FL alone or combined with anti-CD8α or anti-CD4 antibodies according to the protocol described in the Materials and Methods The size of the tumors was determined twice a week for groups of 5 animals ( ^* ) represents tumors of significantly smaller size in the groups of mice injected with FL and not depleted compared with animals treated with the anti-CD8α antibody These data were reproduced twice with similar results

(b) Co-administration of CTLA4lg in vivo Similar experiments were carried out with the mouse antibody CTLA4lg as described in the Materials and Methods ( ^* ) represents tumors significantly larger in the group of animals treated with CTLA4lg in comparison with animals treated with FL only Cos data were reproduced twice with similar results

(c) Co-administration of anti-p40 - mlL-12 monoclonal antibody in vivo Similar experiments were carried out with the anti-p40 - mlL-12 antibody as described in the Materials and Methods No significant blocking of the antitumor activity was observed These data were duplicated twice with similar results

(d) Co-administration of anti-IFN-γ monoclonal antibody in vivo Similar experiments were carried out with an anti-IFN-γ antibody (*) represents tumors of significantly larger size in the groups treated with anti -IFN-γ by comparison with animals treated with FL alone These data were reproduced twice with similar results

FIGURE 8 Adoptive transfer of dendntic cells of splenic origin into B6-nude mice carrying AK7 tumors prophylactic and therapeutic effects 8a From 2 to 5 million immature dendntic cells were injected by direct subcutaneous intratumoral route in nude B6 mice carrying AK7 tumors on day 1 (prophylaxis) The injections were carried out twice a week for 15 days Growth was checked and compared with a group of animals treated with PBS (five mice per group) according to the t-student test. Significant results at 95% are indicated by a (*)

8b Idem but the dendntic cells were injected into AK7 tumors established for 20 days (in therapy)

FIGURE 9 Activation of purified human NK cells from PBL from healthy or sick donors by immature dendntic cells triggered in the presence of fibroblasts (L-cells) or supernatant (SN L cells) 9a Measurement of IFNγ secretion The results are expressed in IU / ml, repeated in at least three separate experiments 9b Measurement of the cytolysis of tumor targets by assaying chromium released (tDC = Dendritic cell triggered (or "tπggered"))

FIGURE 10 The exosomes of dendntic cells muπnes (DEXm) activate the cells NK muπnes 10 (a) Measurement of the production of interferon gamma, 10 (b) measurement of the cytolysis of the cells YAC1, 10 (c) measurement of the rate of survival NK cells, 10 (d) measurement of the average size of tumors in vivo Dex BM-DC dexosomes produced from dendntic cells derived from bone marrow Ratio AND ratio of cells effectπces on target cells SN BM-DC direct cell supernatant bone marrow derivatives

FIGURE 11 Human dendntic cell exosomes (DEXh) activate the resting furnished NK cells Dex MD-DC dexosomes produced from dendntic cells derived from SN monocytes MD-DC direct supernatant from dendntic cells derived from monocytes

IGURE 12 "Knock-" mouse dendntic cell (DEXm) exosomes

Out "for the beta-2-mcroglobulin gene (12a) or for MHC class II molecules (12b) activate NK cells with B6wt B6 wild type β2m mice - / -" Knock-Out "mice for beta-2-mcroglobulin gene Cl II - / - "Knock-Out" mouse for MHC class II molecules FIGURE 13 Relative activities of dendntic cells and the dexosomes they produce to stimulate NK cells 13 (a) Comparative study of the activity of dendntic cells produced in low or high dose of interleukιne-4, and dexosomes that 13 (b) Comparative study of the activity of triggered and non-triggered dendntic cells, and of the dexosomes they produce

MATERIALS AND METHODS

1 ANIMALS

The female mice C57-BL / 6 (B6) and BALB / c scid / scid (SCID) were obtained from the Center d'Elevage Janvier (Le Genest St-lsle, France) The female mice C57 / BL / 6- bg / bg (B6-beιge) were purchased from Harlan UK Limited (Oxon, England) The female mice C57-BL / 6-B6-nude (B6-nude) were obtained from the Mollegaard A / S Research and Breeding Center (Skensved, Denmark) The female and male C57BL / 6 Rag2 - / - (B6-Rag - / -) mice were purchased from the Center for Development of Advanced Techniques of the National Center for Scientific Research (Orléans, France) The mice Beige SCID and B6-nude were maintained under pathogen-free conditions The mice were grouped by age (8 to 10 weeks) at the start of each experiment

2 CELL CULTURES

AK7 cells, provided by A Kane (Brown University, Providence, Rhode Island), are a line of mesothelioma muπn generated by intra-pertoneal injection of crocidolite asbestos fiber in B6 mice. This cell line was maintained in DMEM supplemented medium. with 10% bovine-fetal serum inactivated by heat treatment, 10 mM sodium pyruvate, 2 mM L-glutamine, 100 IU / ml of penicillin, and 100 μg / ml of streptomycin Tumor cell lines have were maintained in vitro, for a period not exceeding one month before the in vivo experiments. The lines of dendntic cells supplied are maintained in an IMDM medium containing 10% of inactivated fetal bovine serum, 2 rnM of L-glutamine, 50 μM of 2- βME, 100 IU / ml of penicillin, and 100 μg / ml of streptomycin (IMDM medium). To induce cell maturation, TNF-α from recombinant mice (R&D Systems, Abiπgdon, UK) is added at a concentration of 10 ng / ml for 24 hours. maturation can also be induced by incubation of cells in the presence of LPS (1-10 μg / ml) The cell line YAC-1 is a lymphoma line in an A / Sn context very sensitive to NK cells P815 cells are cells mastocytoma in DBA / 2 context resistant to NK cells All culture media and reagents were obtained from GIBCO BRL (Life Technologies, Merelbeke, Belgium)

3 GENERATION AND CULTURES OF DENDRITIC CELLS DERIVED FROM BONE MARROW

Munnite dendntic cells come from the differentiation of precursor cells from the bone marrow cultured in the presence of GM-CSF and IL-4 for 6 days in RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine, essential amino acids, penicillin, streptomycin and β-2ME These cells were obtained according to a protocol described by Mayordomo et al (1996) Briefly, the bone marrow is extracted from shins and femurs, depleted in lymphocytes and macrophages, and spread on 24-well culture plates (0.25 10 ⁶ cells / ml) in RPMI 1640 medium defined above supplemented with rm IL-4 and rm GM-CSF (1000 IU / ml of each) Au day 3, the cells with little or no adhesion are harvested and spread on 24-puιts culture plates (0.3 10 ⁶ cells / ml) for 3 additional days of culture with the same new medium The dendritic cells thus obtained have the phenotype expected on s dendntic cells thus obtained, are immature cells They can be induced for maturation in culture combining ιnterleukιne-4 and GM-CSF with TNF-α (10 ng / ml) and / or LPS (1-10 μg / ml) The cells are used 24/48 hours after the maturation stimulus for co-culture Autologous and allogeneic dendntic cells have been used for co-culture with the same efficacy In addition, mature and immature dendntic cells can also be used interchangeably

4. TRIGGERING DENDRITIC CELLS Dendntic cells can be triggered according to different protocols. One of the methods used in the examples comprises the coculture of dendntic cells (mature or immature) in the presence of dividing fibroblastic cells (in particular cells of the line L-929 or NIH 3T3) The coculture is generally maintained for 24- 48 hours, tDCs which can be obtained from approximately 20 hours of coculture According to another method, the dendritic cells are incubated in a triggering medium, advantageously comprising culture supernatant of dividing fibroblast cells (in particular of cells of line L-929, NIH3T3 or MRC5, primary culture of human pulmonary fibroblasts) In the examples which follow, the supernatant used was diluted to a third, and the cultures were carried out in a medium comprising IL-4 and GM-CSF Another triggering experiment was carried out using as triggering medium, a medium comprising culture supernatant of tumor cells (mastocytoma)

5 GENERATION OF NK CELLS

The NK cells are obtained in the rest state from the spleens of SCID or Rag2 mice - / - after a plastic adhesion step for 2 to 3 hours at 37 ° C. Briefly, the spleens of B6-Rag- / mice - or SCID mice were dissociated in complete RPMI 1640 medium as defined above to generate cell cultures in suspension After lysis of the erythrocytes, the cells were washed once and spread (2.5 × 10 ⁶ cells / ml ) for 2 to 3 hours at 37 ° C. then the non-adherent cells were harvested and counted These cells are essentially composed of NK cells at rest

6 CO-CULTURE NK / DC

For the co-cultures, the immature or mature, autologous or allogenic dendntic cells, triggered or not, were harvested, washed 3 times and added in different concentration ratios to NK cells at rest, freshly isolated (1 10 ⁶ NK cells ml) in 96-well plates with U-bottom The cells incubated individually (dendntic cells or NK cells) are spread at similar concentrations as controls

7 INTERFERON γ SECRETION TEST

The supernatants of dendntic cell / NK cell co-cultures are collected and, where appropriate, stored at -70 ° C. The assays for the release of interferon γ release are carried out using commercial ELISA Kits (Genzyme Corp. Cambridge, United States ) The detection sensitivity of the tests used is close to 5 pg / ml Dose effects are achieved by varying the ratio of dendritic cells to NK cells 8 NK CELL CYTOTOXICITY TEST

The in vitro cytotoxicity of NK cells is evaluated according to conventional methods of the release of chromium 51 from targets marked sensitive NK (YAC-1) or resistant NK (P815)

More specifically, cells from NK / CD co-cultures or from separate cultures incubated for 40 to 72 hours, were collected, labeled with Trypan Blue to exclude lymphocytes and dendntic cells, and counted and used as effectπces cells in a test. of cytotoxicity using YAC-1 and P815 cells as target cells The target cells were pre-incubated for 1 to 2 hours at 37 ° with Na ⁵¹ Cr04 (100 μCι / 10 ⁶ cells, New England Nuclear) washed one times, incubated in RPMI 1640 medium supplemented with 5% of inactivated fetal bovine serum, for 1 hour at 37 ° C. The cells are then washed 3 times and spread at 2 × 10 ³ cells per well, in 96-well microtiter plates having a V-bottom The effectπces and target cells were mixed at different effector / target ratios in a total volume of 0.2 ml and incubated for 4 hours at 37 ° C. The degree of lysis of the target cells was measured by counting 0 1 ml of supernatant transferred to a Luma plate Spontaneous release was determined from wells containing target cells labeled alone and the maximum release of chromium 51 was determined by addition of 2% of ketπmide Specific cytotoxicity a was calculated as follows percentage of chromium ⁵¹ release = 100 x (experimental cost cpm - cost of spontaneous release) / (cost of maximum release cpm - cost cpm of spontaneous release)

For the m vivo tests, the spleens of 2 or 3 mice carrying AK7 cells or devoid of tumors were removed after 20 days of treatment with the compound FL or with a phosphate buffer (PBS) Suspensions of spienocyte cells depleted in erythrocytes by osmotic lysis were immediately used as effected cells in a cytotoxicity test using the YAC-1 and P815 cells as targets as described above

9 IN VIVO MOUSE TREATMENT WITH E COMPOUND FL

Mice are inoculated intradermally into the right flank with the minimum tumor dose of AK7 cells (3 10 ⁶ cells) in a volume of 0.1 ml of PBS.

The FL compound derived from CHO cells was supplied by Immunex Corp (Seattle, USA, Lynch et al, Nature Médecine 3 625, 1997). This cytokine was used in diluted form in PBS to 100 μg per ml AK7 tumors established on day 20 (approximately 20 mm ² in diameter) were treated with a single daily injection (subcutaneous injection in the left flank) either of FL or PBS (10 μg) in a total volume of 0.1 ml for 20 consecutive days. The mice were checked for tumor growth twice a week and the average size of the tumors was illustrated by measuring two perpendicular diameters in millimeters using a caliper. The tumor growth rates were determined by reporting the size of the tumors ( mm ² ) relative to the time after day 1 of treatment with LF All studies were carried out at least 4 times with groups of 5 animals

10 EXPERIENCES OF DEPLETION BY ANTIBODIES IN VIVO Antibodies anti-CD8α (clone YTS 191 1.2, rat lgG2a), anti-CD4 (clone YTS 169 4 2 1, rat lgG2a) and antι-NK1 1 (clone PK136, lgG2 mice) were used in the depletion experiments The hybπdomes were cultivated in athymic mice and the ascites fluids were harvested and purified according to the techniques described above by precipitation with caprylic acid then with ammonium sulphate, dialyses against PBS, then adjusted to 1 mg / ml (McKinney, 1987) Unless contraindicated in the legends of the figures, depletion began on day 1 of treatment with FL of B6 mice, 200 to 300 μg of monoclonal antibody are injected with intraperitoneal route for 3 consecutive days, then every two days during the FL treatment After the FL treatment, the monoclonal antibodies are administered twice at 4 days apart Experiments carried out in parallel p ar flow cytometry by means of fluorescent antibodies made it possible to confirm that the depletions observed are greater than 99% for the targeted cell subpopulations, on day 9 and on day 20 of the FL treatment For the B7 blocking experiments, the mice were injected intraperitoneally with 200 μg of CTLA4lg mouse fusion protein (supplied by L Adoπni, Hoffman La Roche, Italy) 2 days apart between day 10 and day 18 of FL treatment For studies for neutralizing endogenous IL-12, the purified antibody antι-p40 IL-12 (clone C-17-8, rat lgG2a), was injected intraperitoneally into individual αoses of 300 μg at 3 days d between day 5 and day 17 of LF treatment For studies to neutralize endogenous interferon γ, 300 μg of anti-interferon γ antibody (hamster IgG) were in _j ected mice intraperitoneally on days 10, 15 and 20 of the administration of FL All the experiments were carried out with 5 mice per group and at least twice

1 1 ADOPTIVE TRANSFER OF DENDRITIC CELLS TO MICE

B6-nude or SCID mice carrying an AK7 tumor on day 1 or day 20 were injected intratumorally subcutaneously twice a week, with 2 to 5 × 10 ⁶ immature dendntic cells for two weeks Five mice per group were treated Tumor growth was monitored as previously described

12 STATISTICAL DATA ANALYSIS

The exact Fisher method was used to interpret the significance of the differences between the different experimental groups (presented as a mean with standard deviation). To interpret passive transfer experiments of dendntic cells, 2 groups were compared using the t- Student test The significance of 95% of the results is represented for each individual experience

EXAMPLES

The results presented in the following examples demonstrate in particular

1) that the coculture of dendntic cells and of purified NK cells leads to the survival of mature dendntic cells and of NK cells and the rapid activation of NK cells, in other words the dendritic cell, in the absence of any other known, can make it possible to make an NK cell highly killer and secrete of IFNγ against its sensitive natural NK targets, without addition of cytokines and without demonstrating detectable levels of secretion of IL-2, IL-12 in these cocultures,

2) that the adoptive transfer of dendntic cells or the direct in vivo expansion of dendntic cells leads to significant antitumor effects due to the activation of NK

EXAMPLE 1 The dendntic cells stimulate the cytolytic activity and the production of interferon γ by the NK cells in vitro This example illustrates the properties of dendntic cells at different stages of differentiation to activate NK cells at rest in vitro Cytotoxic activity, production of interferon γ and proliferation were tested

In a series of experiments, dendntic cells freshly derived from syngeneic bone marrow were cultured in the presence of ιnterleukιne-4 and GM-CSF and maintained in the immature state (see materials and methods) The immature dendntic cells were collected, intensively washed and co-incubated in an initial ratio of approximately 1 1 with non-adherent mononuclear cells obtained from splenocytes freshly harvested from syngeneic SCID BALB / c mice, in complete medium in the absence of cytokine One third of these nonadherent splenocytes are positively labeled with the monoclonal antibody Dx5 or with the monoclonal antibody antι-NK1 1 After co-culture for 40 to 72 hours, the NK cells are counted and tested in a cytotoxicity test by measurement of the release of chromium 51 for 4 hours against YAC-1 and P815 cells at different effector / target ratios

The results obtained are presented in FIG. 1 and show that these dendntic cells induce the activation of the cytolytic activity of NK cells. In particular, these results show

- that the co-culture of the two cell types makes it possible to harvest viable NK cells, and

- that when the NK cells and the dendntic cells are co-incubated at a ratio of 0.1 to 0.3 1, a specific lysis of 20 to 50% of the YAC-1 cells is obtained at a ratio of effective cells / target cells from 25 to 100 1 P815 cells, which are NK insensitive, are not lysed under the conditions tested and neither of the two cell populations (NK cells and dendntic cells) cultivated separately at similar concentrations induces significant lysis of YAC cells -1 (Figure 2)

In another series of experiments, a spleen dendritic cell line was used. This line was maintained under culture conditions allowing long-term growth of cells in the immature stage and their triggering. This line was then induced to mature. in the presence of TNFα or LPS for 24 hours Matured cells exhibit significant morphological changes as described previously by Wiπzler et al. The aggregates are no longer adherent and analyzes by FACS reveal an important expression of the molecules CD11c, l-Ab, B7.2, and CD40. These cells mature dendntiques were tested under the same conditions as those described above, by coincubation for 40 to 48 hours with NK cells at rest freshly harvested in B6-Rag mice - / - at different ratios The results obtained are presented on the Figure 2 These results show that - the co-culture of the two cell types makes it possible to harvest viable NK cells in the wells significantly higher than in a separate NK cell culture (10 to 30% vs 2 to 5%)

- when the NK cells and the dendntic cells are co-incubated at a ratio of 0.1 to 0.3 1, a specific lysis of 40 to 60% of the YAC-1 cells is obtained at a target cell / effective cell ratio of 25 1 P815 cells which are insensitive NK, were not lysed under all the conditions tested Neither of the two cell populations (NK cells and dendntic cells) cultured separately in culture medium at similar concentrations did not induce lysis significant of YAC-1 cells (Figure 2a) Lysis of other MHC class I weak positive tumor cells, such as MCA205, MCA101, TS / A or AK7 was also observed in the presence of activated NK cells (results not shown)

The results also show that the co-culture supernatant NK-DC contains significant levels of interferon γ which decrease as a function of the number of stimulating dendntic cells (FIG. 2b) On the other hand, interferon γ was not detectable in the supernatant of dendntic cells or of NK cells cultivated separately No significant proliferation was observed under these co-culture conditions as determined by the incorporation of thymidine after a co-culture of 40 to 70 hours

These results therefore illustrate the capacity of immature and mature dendntic cells to stimulate the activity of NK cells both for the production of interferon γ and for the specific lysis activity against tumor cells, sometimes exceeding the KIR effect.

In a similar manner to the previous experiments, it was also demonstrated that the allogeneic dendntic cells were capable of stimulating the activation of NK cells 3rιόvemcnt, allogenic splenocytes nonadherent of SCID BALB / c mice were activated by co-culture of 40 at 48 hours in the presence of mature dendntic cells at an NK.DC ratio of

0.1 to 0.3 1 The results are presented in FIGS. 2a and 2b and show that these Allogeneic dendntic cells activate both the NK cell lytic properties and the production of γ interferon

All of these results clearly show that mature or immature, autologous or allogeneic dendntic cells, whether dendntic cells derived from bone marrow or spleen or established lines of dendntic cells, are capable, when they are triggered, to effectively activate by co-culture the lytic activity of NK cells as well as the production of interferon γ by NK cells

EXAMPLE 2 Stimulation of NK cells by dendntic cells involves direct cell contact

Mature dendntic cells are known to secrete different cytokines such as IL-12, IL-15, IFNα / β or TNFα which could be responsible for the activation of NK cells. A "Transwell" culture system has was used to determine the possible implication of soluble factors in the activation of NK cells by dendntic cells The results presented in FIG. 3 show that the cytolytic activity and the production of interferon γ by NK cells in response to cells dendntiques is detected only in the case where the NK cells are co-incubated in contact with the dendntique cells but not when the cells are separated by a porous membrane This & results therefore show that the activation of NK cells by the dendntique cells requires direct cell / cell contact or interaction, and that this activation involves a co-stimulation factor, present in the membrane of dendntic cells

EXAMPLE 3 The expansion of white blood cells of the dendritic line by the compound FL is accompanied by the regression of an established class 1 negative tumor in B6-nude mice or in Rag2 B6 - / -

AK7 cells represent a line of syngeneic mesothelioma of weakly immunogenic B6 mice which spontaneously infiltrate the peroneal cavity after prolonged establishment in the abdominal flank The AK7 tumor thus obtained expresses very low levels of class I molecules in vitro Treatment with the compound FL of AK7 tumors established on day 20 in B6-nude mice induces a transient suppression of tumor growth and finally a significant growth retardation m vivo (see FIG. 4) The FL compound does not, however, have any effect direct cytotoxic on AK7 cells in vitro These anti-tumor effects m vivo begin to be observed on day 10 of treatment with LF when splenomegaly and adenomegaly, attributable to the expansion of white blood cells of the dendritic line, have been observed The kinetics of tumor growth resume a normal rhythm 5 to 10 days after stopping the FL treatment, when the number of dendntic cells decreases The anti-tumor effects induced by FL observed in the B6-nude or Rag2 B6 - / - mice were also pronounced than those reported in immunocompetent mice (Figures 5b), stressing that neither T cells nor B cells are involved in tumor growth retardation

These results therefore demonstrate that the administration of a growth factor of dendntic cells induces a regression of negative class I tumors. These results also show that this effect is not due to the activity of T or B cells.

EXAMPLE 4 The anti-tumor effects mediated by the compound FL are not observed in beige mice and are blocked by the administration of a monoclonal antibody aπtι-NK1 1 in immunocompetent mice carrying the tumor.

In order to more precisely determine the mechanisms of the anti-tumor effects induced by FL, B6-Belgian mice carrying the AK7 tumor were treated with FL according to a similar therapeutic regime.

The beige B6 mice present mesotheliomas which grow progressively until death, despite administration of FL (Figure 5a) In order to determine more precisely the involvement of NK cells in the tumor activity induced by FL, B6 mice immunocompetent were depleted using monoclonal antibodies antι-NK1 1 As the results shown in FIG. 5b show, this depletion is necessary and sufficient to completely cancel the anti-tumor effects induced by FL Consequently, these results show that the NK cells play a necessary and sufficient critical role in the effectiveness of the FL compound as an anti-tumor agent

EXAMPLE 5 Compound FL significantly increases the spleen NK activity in vivo

This example illustrates the capacity of the compound FL to increase the activity of NK cells in vivo. The absolute number of positively labeled NK cells with the monoclonal antibody antι-NK1 1 by FACS analysis in the splenocytes and the mononuclear cells of the lymph nodes were slightly increased from 3 to 5 times in the mice presenting the tumors and in the mice without tumor The splenocytes collected on day 20 of B6 mice treated with the compound FL or with a saline solution, in mice having or not showing AK7 tumors, were tested for spontaneous cytolytic activity against YAC-1 cells in vitro in a chromium 51 release test on a 4 hour period A significant increase in NK activity but not in the production of interferon γ has been shown in various experiments carried out both with nude or immunocompetent mice, with no significant effect attributable to the presence of the tumor itself. same (Figure 6) No lysis was observed against P815 cells In addition, the cytokine levels for interferon γ and TNFα in serum or culture supernatants of mononuclear cells of spleen lymph nodes were not statistically different under all conditions tested and no detectable level was observed for IL-1β and IL-12 Ces results therefore show that treatment with the compound FL is associated with an increase in NK activity in the spleen in vivo

EXAMPLE 6 Depletion of Nude mice by administration of anti-CD8α monoclonal antibody or of CTLA4-lg significantly blocks the anti-tumor effects dependent on NK cells

This example describes the role of the bous -populations of myeloid or lymphoid dendntic cells induced by the FL compound in the peripheral increase in cytolytic NK activity and in NK-dependent anti-tumor effects Since the lymphoid dendntic cells have been described as secreting IL-12 in response to SAC + IFNγ stimulation in vitro and insofar as IL-12 is a stimulating factor for NK cells, selective depletion of CD8α positive lymphoid dendritic cells (DEC205 +) using monoclonal antibody antι-CD8α was undertaken in B6-nude mice The injection of the depleting monoclonal antibody was started as soon as differentiated dendntic cells in the presence of FL (day 0 FL) appeared and continued for up to 10 days after discontinuation of FL treatment at high doses The CD8α molecule is not expressed on mouse NK cells in B6-nude mice, which ren d thus the depletion targeted towards the subpopulations of lymphoid dendntic cells Mice carrying tumors AK7 depleted with the anti-CD8α monoclonal antibody respond significantly less to FL treatment than non-depleted control mice or mice injected with an anti-CD4 antibody (FIG. 7a) However, the FL compound remains effective in mice having received FL and depleted for CD8α positive cells in comparison with animals not treated with the FL compound, emphasizing that lymphoid dendntic cells are only partially involved in anti-tumor effects dependent on NK cells The small population of myeloid dendntic cells expressing the CD4 marker seems not to participate in these anti-tumor effects (FIG. 7a) To further confirm the critical role of dendntic cells in the anti-tumor effects dependent on NK cells, co-stimulation molecules B7 which are known to be involved in the effectπce phase of recognition by NK cells, were targeted using a systemic injection of the muπne fusion protein CTLA4-lg During the complete period of administration of CTLA4-lg, a statistically significant loss of efficacy of FL on the suppression of tumor growth was observed (FIG. 7b). an injection of human IgG in the control experiment did not alter the efficiency of FL in vivo It should be emphasized that the AK7 tumor does not express B7 molecules in vitro Overall, these data show that the cells B7 positive and / or dendntic cells are essential in NK cell dependent anti-tumor effects obtained by FL growth factor

EXAMPLE 7 Interleukme 12 is not involved in the anti-tumor effects NK induced by FL The cytokines IL-12 and IFN-γ are T cytokines of type Th1 which are known to stimulate NK activity in vivo In addition , as described in the previous example, the subpopulation of lymphoid dendntic cells capable of secreting IL-12 is important for the anti-tumor effect observed Although the levels of IL-12 in the sera or supernatants from lymph nodes or spleens derived from mononuclear cells, were undetectable in animals treated with! ^" -L, souns were injected with the neutralizing antibody antι-p40 IL-12 from day 5 until day 20 of treatment with FL No inhibitory effect of these monoclonal antibodies on the anti-tumor effects induced by FL n was observed in vivo (FIG. 7c) Neutralization of the interferon γ in vivo makes it possible, on the other hand, to temporarily block the anti- tumors mediated by FL, which implies that the cytotoxicity of NK is mediated by IFNγ because the mesothelioma is IFNγ sensitive (FIG. 7d) Other results obtained confirm these observations In particular, (i) knockout mice for the type-1 interferon receptor always have NK cells activatable by the dendntic cells according to the invention, (ii) the supernatants of these cocultures do not proliferate CTLL2, which attests to the absence of IL -2 or IL-15 produced under these conditions, and (H ^' I) the antibody aπti-IFNγ does not interfere much with the activity of the FL compound in vivo

EXAMPLE 8 The adoptive transfer of dendntic cells at the start of tumor establishment or during tumor growth at D20 in SCID mice affects tumor growth

In order to definitively rule out any indirect event likely to account for the increase in NK activity independent of the developed population of dendntic cells and so as to formally test a direct vivo effect of dialogue between dendntic cells and NK cells from B6-nude or SCID mice carrying AK7 tumors, known to exhibit strong NK activity, were passively transferred with 2 to 5 million immature dendntic cells by subcutaneous injection twice a week for two weeks from D1 or from D20 AK7 The results are presented in FIGS. 8a and 8b and show a significant delay in tumor growth in the group of mice treated with dendntic cells in comparison with the control group. These results clearly demonstrate the role of dendntic cells in stimulating NK cell activity in vivo

EXAMPLE 9 Triggering of immature dendntic cells by xenogenic trigger medium

This example illustrates the fact that immature dendntic cells can be triggered in a xenogenic medium (in the presence of a factor), and that the trigger does not modify their stage of maturity

5x10 ⁶ immature CD derived from monocytes (MD-CD) in IL ~ 4 + GM-CSF (CM) for 8 days were cultured, for 24-48 hours (from D8 to D10) in the presence ^* - either a medium composed of CM (2/3 of the final medium) and a triggering medium (1/3 of the final medium) corresponding to the supernatant of 10 × 10 ⁶ L-929 fibroblasts cultured for 48 hours,

- either in a CM medium, in the presence of 1x10 ⁶ irradiated L-929 fibroblasts (5000 rads)

After 48 hours of incubation, the MD-DCs were recovered and put in coculture alone or in the presence of allogenic human NK cells, purified by negative immuπoselection from the blood of healthy donor (culture ratio 2 DC for 1 NK, concentration 0.3 × 10 ⁶ NK / ml) The DC / NK or DC alone or NK alone coculture supernatant was then evaluated by ELISA to assay for IFNγ. At the same time, the cells from the cocultures were recovered, listed and placed on targets. chromed 51 Cr to assess their lytic power over 4 hours The results obtained are presented in Figure 9 These results clearly show the strong capacity of the triggered immature dendntic cells to activate the secretion of IFNγ and the lytic activity of NK cells at rest

EXAMPLE 10 Activation of NK Cells by a Preparation Derived from Dendntic Cells

This example illustrates the fact that preparations derived from dendntic cells can be used to activate NK cells. In particular, this example shows that membrane vesicles (or dexosomes) produced from dendntic cells activate NK cells.

In this example, the human MD-DC cells were cultured from the adherent fraction of monocytes from healthy subjects or carriers of metastatic tumors (melanomas, myelomas, lymphomas) in culture medium such as AIMV + PeniStrepto + L-Glutamπe + 10% FCS + 1000IU / ml of IL-4 and GM-CSF The medium is changed on D5 or D6 then the supernatants of these MD-DC incubated at 37 ° C, 5% C02 for 24-48 hours, are passed through a 0.2μm filter then ultracentnfuged to 70,000 g to isolate dexosomes The munnes BM-DC were cultured and triggered as described in the materials and methods (points 3 and 4) In addition, the spinal precursors were also and alternately cultivated in GM-CSF alone at 1000 lU / ml The DCs were cultivated until D6, and the culture supernatants from which the dexosomes come (DEXm) are accumulated from D3 to D6 They are then recovered according to the procedure described by example in patent applications FR9709007 and FR9801437, that is to say by differential ultracentπfugations

Munne and human NK cells were collected as described in the materials and methods (point 5). For bringing together NK cells and dexosomes, the NK cells of SCID / BALBc mouse spleens were incubated in 96-well plates. a conical bottom, at a concentration of 1.5 million / ml (these splenocytes contain 30% Dx5 + NK) Then, the dexosomes were added, at a rate of 10-20 μg of exosomal proteins (Bradford test) per well, and the cells were recovered at 18-20 hours to carry out the cytotoxicity (against YAC-1 cells) and gamma interferon production tests (supernatant tested in ELISA IFNg munn) The cytotoxicity and interferon assay tests were were carried out as described in the materials and methods (points 7 and 8)

The results obtained are presented in FIGS. 10 to 13 These results clearly show an activation of the NK cells by the dexosomes of dendntic cells, activation particularly pronounced by the dexosomes produced from immature dendntic cells and not triggered In addition, the activation observed crosses the species barrier since human dexosomes activate muNne NK cells More particularly, Figure 10 (a) shows the production of IFNγ munn by

NK of SCID BALB / c mice (splenocytes comprising 30 to 40% of Dx5 + cells, that is to say of NK cells) stimulated by an exosome preparation originating from dendntic munne cells cultured in IL-4 + GM-CSF (BM-DC), at a dose of the order of 20 μg / ml of Dx5 + NK At this dose, the exosomes also increase the basic cytotoxicity of the NKs at rest (Figure 10b) as well as their survival in vitro (Figure 10c) , whereas, in most of these cases, the direct supernatant of BM-DC has no major effect on the activation of NK (FIG. 10c) The results presented in FIG. 10d further confirm this activity in vivo , since they show a decrease in the average size of AK7 tumors in "nude" mice after administration of dexosomes These results therefore show that the exosomes of immature dendntic cells stimulate the activity of NK They also show that the level of activation is influenced by the concentration of dexosomes, and that doses greater than or equal to approximately 10 μg / 10 ⁶ NK cells are preferred for activation in vitro or ex vivo The results presented in FIG. 11 further confirm these observations on dexosomes of human dendntic cells. In this experiment, the MD-DCs were cultured in IL-4 + GM-CSF for approximately 9 days. The exosomes (DEXh) secreted by these cells, which are in an essentially immature state, were recovered from D7 to D9 or from D6 to D8 These human dexosomes (DEXh) were incubated under the conditions described above in the presence of NK cells, munnes, at doses ranging from 20 to 40 μg / million NK cells The results obtained show a stimulation of the production of IFNγ, demonstrating the activation of NK cells

Furthermore, the results presented in FIG. 12 show that the exosomes of CD munnes coming from deficient mice ("Knock Out") for the beta2-microglobulin gene (classic or related MHC class I molecule) or for the molecules of MHC class II retain their ability to stimulate NK at rest for the production of IFNγ These results (i) confirm the stimulatory activity of dexosomes and (u) indicate that the stimulating factor does not seem to be a molecule of MHC class I (unlike certain elements of the family of KIRs-Killer inhibitory receptor, small, or KIRI-killer inhibitory receptor, long) The results obtained show in particular that the production of IFN-γ by NK stimulated with CD dexosomes from mouse beta2mιcroglobulιne - / - KO (Figure 12a) or class II KO (Figure 12b) are strong NK stimulation was also observed using whole dendntic cells

Finally, FIG. 13 shows a certain correlation between the stimulatory activity of the dendntic cells and the exosomes which they secrete. Thus, the present results show that highly active dendntic cells (for example mature and, if necessary triggered) secrete dexosomes. more weakly active On the other hand, dendntic cells exhibiting a weaker activity (for example immature and non-triggered dendntic cells) secrete very active dexosomes These elements therefore suggest a transfer, depending on the state of the dendntic cells, of the stimulation factor from the internal membrane vesicles up to the external plasma membrane More particularly, FIG. 13a shows that the exosomes of CD cultured in the presence of GM-CSF and low doses of ιnterleukιne-4 (1x) significantly stimulate the NKs than their counterparts from CDs grown in the presence of GM-CSF and high doses (5x ) of IL-4, while the situation is reversed for the CDs from which they are derived Similarly, Figure 13b shows that muπns exosomes secreted by triggered BM-DC cells (supernatant of L929) significantly stimulate NKs less than their counterparts from non-triggered BM-DCs, and that the situation is reversed for CD from which they are derived Comparable results have been obtained with human dendntic cells

All of these results therefore show (i) that the dexosomes are capable of stimulating the activity of NK cells at rest, (ii) that the most active dexosomes seem to be produced by less active dendntic cells, ie ie immature (and possibly not triggered), (iii) that activation by dexosomes crosses the species barrier, (iv) that the stimulation factor responsible for the activation of NK is on the surface of DEX and presented to NK directly, with or without costimulation (another membrane factor or cytokine) and (v) that the stimulation factor does not seem to be a MHC class I molecule These results therefore support the use of dexosomes for the activation of NK in vitro, ex vivo or in vivo

Claims

1 Method for activating natural killer cells (NK) comprising bringing NK cells into contact with dendntic cells or a preparation derived from dendntic cells, in vitro or ex vivo

2 Method according to claim 1 characterized in that the dendntic cells are mature dendntic cells

3 Method according to claim 1 characterized in that the dendntic cells are immature dendntic cells 4 Method according to one of claims 1 to 3 characterized in that the dendntic and NK cells are autologous

5 Method according to one of claims 1 to 3 characterized in that the dendntic and NK cells are allogenic

6 Method according to one of claims 1 to 5 characterized in that the dendntic cells are triggered cells

7 Method according to any one of the preceding claims, characterized in that the ratio of NK cells / dendntic cells is between 0.01 and 10, preferably between 0.05 and 5

8 Co-culture of dendntic cells and NK cells 9 Composition comprising activated or resting NK cells and autologous dendntic cells

10 Composition comprising NK cells activated by the method of claim 1

11 Use of dendntic cells or of a preparation derived from dendntic cells, for the activation of natural killer cells in vitro or ex vivo

12 Use of dendntic cells or of a preparation derived from dendntic cells, for the preparation of a composition intended to activate natural killer cells in vivo

13 Use of dendntic cells or of a preparation derived from dendntic cells, for the preparation of a composition intended to activate the cytolytic activity of natural killer cells in vivo

14 Use of dendntic cells or of a preparation derived from dendntic cells, for the preparation of a composition intended to increase the production of IFNγ by NK cells in vivo 15 Use according to claims 11 to 14 characterized in that the dendntic cells are mature dendntic cells

16 Use according to claims 11 to 14 characterized in that the dendntic cells are immature dendntic cells 17 Use according to claims 11 to 16 characterized in that the dendntic cells are triggered dendntic cells

18 Use according to claims 11 to 17 characterized in that the dendntic cells are sensitized to one or more antigens

19 Use of a dendntic cell growth factor for the preparation of a composition intended to activate natural killer cells in vivo

20 Use according to claim 19 characterized in that the growth factor is the compound FL

21 Composition comprising a stimulating factor for NK cells derived from dendntic cells 22 Composition comprising a compound capable of interfering with the interaction between dendntic cells and NK cells

23 Use of a factor according to claim 21 for the preparation of a composition intended to activate natural killer cells in vivo

24 Use of a compound according to claim 22 for the preparation of a composition intended to negatively control the activity of natural killer cells in vivo

25 Use of dendntic cells or a composition according to claims 9 or 10 for the preparation of a medicament intended for the treatment of tumors 26 Use of dendntic cells or a composition according to claims 9 or 10 for the preparation of a medicine to treat infected cells

27 Use of a composition according to claim 21 for the preparation of a medicament intended for the treatment of tumors or infected cells 28 Use of a composition according to claim 22 for the preparation of a medicament intended for the preventive treatment of rejection of transplant or graft versus host disease (GVHD) 29 Use of a dendntic cell growth factor for the preparation of a composition intended for the treatment of negative or weakly positive class I tumors by increasing NK activity in situ

Use of a dendntic cell membrane factor for the preparation of a composition intended to increase the survival or proliferation of NK lymphocyte subpopulations

31 Use of a membrane factor of NK cells for the preparation of a composition intended to increase the survival of mature dendntic cells

32 Use of dendntic cells with a tolerogenic phenotype or a product derived from tolerogenic dendntic cells for the preparation of a composition intended to inhibit the activation of NK cells

33 Method for treating dendntic cells comprising contacting dendntic cells with a triggering medium, making it possible to improve (or provoke) their capacity for stimulating NK cells 34 Composition comprising at least one factor for triggering dendntic cells and a growth factor for dendntic cells, for their simultaneous, separate or time-separated use

35 Composition comprising triggered dendntic cells, in particular human 36 Triggering medium for dendntic cells, characterized in that it comprises cells of the extracellular matrix, a preparation derived from such cells or a factor derived from such cells

37 A method according to claim 1 characterized in that the preparation derived from dendntic cells comprises membrane vesicles 38 A method according to claim 37, characterized in that they are membrane vesicles produced from immature dendntic cells

39 Use according to one of claims 11 to 14, characterized in that the preparation derived from dendntic cells comprises membrane vesicles

40 Use of membrane vesicles produced from dendntic cells for the preparation of a composition for stimulating cell activity

NK, in vitro, ex vivo or in vivo

41 Method for identifying and / or preparing a factor for stimulating NK cells, comprising contacting a biological material comprising a membrane fraction and a population of NK cells, demonstrating a activation of NK cells, and isolation of activation factor from biological material

42 Method for identifying a compound capable of inhibiting the activation of NK cells, comprising the incubation of a test compound or of a composition comprising one or more test compounds, with NK cells at rest and cells dendntiques, or exosomes, or an NK stimulating factor as defined in claim 21, measuring the activation of NK cells, and selecting compounds / compositions capable of inhibiting (ie, reducing) activation of NK cells