Method for Monitoring the Immunological Profile of a Subject

Abstract

The invention relates to a method for monitoring the immunological profile of a subject, comprising measuring the expressions of each member of the group consisting of NKp30a, NKp30b, NKp30c, NKp44b and NKp44c, in a biological sample of said subject, wherein: if the expressions of NKp30a, NKp30b, and NKp44b are the three highest among said group, then said subject has a responsive profile; or if the expressions of NKp30c and NKp44c are the two highest among said group, then said subject has an unresponsive profile.

Claims

1-4. (canceled)

5. Method according to claim 15, wherein said biological sample is a biopsy or a blood sample.

6. Method according to claim 15, wherein the subject is grafted with stem cells; or has undergone a bone marrow transplant.

7. Method according to claim 17, wherein said cancer is selected from the group consisting of melanoma, colon cancer, renal cancer and haematological malignancies.

8. Method according to claim 17, wherein said viral infection is an infection caused by a virus selected from the group consisting of HIV, hepatitis E virus, hepatitis C virus, cytomegalovirus, Epstein-Barr virus and influenza viruses.

9-11. (canceled)

12. Method for converting a sample of NK cells having a responsive profile into NK cells having an unresponsive profile, comprising a step of mixing said sample with a composition comprising TGF- and IL-15.

13. Method according to claim 12, wherein the composition further comprises IL-18.

14. Method according to claim 12, wherein the composition comprises: a) 1.5 to 4 ng/ml of TGF-; b) 5 to 20 ng/ml of IL-15; and c) optionally 5 to 20 ng/ml of IL-18.

15. A method for treating a subject grafted with cells or organ, comprising the following steps: i) measuring the expressions of the group of NKp30a, NKp30b, NKp30c, NKp44b and NKp44c in a biological sample of said subject; ii) treating said subject with an immunosuppressive drug when the expressions of NKp30a, NKp30b, and NKp44b are the three highest among said group.

16. A method for treating a subject grafted with cells or organ, comprising the following steps: i) measuring the expressions of the group of NKp30a, NKp30b, NKp30c, NKp44b and NKp44c in a biological sample of said subject; ii) mixing the biological sample of step i) with a composition comprising TGF-, IL-15 and optionally IL-18 when the expressions of NKp30a, NKp30b, and NKp44b are the three highest among said group; and iii) introducing the mixture obtained at the end of step ii) into the subject.

17. A method for treating a subject suffering from a viral infection or cancer, comprising the following steps: i) measuring the expressions of the group of NKp30a, NKp30b, NKp30c, NKp44b and NKp44c in a biological sample of said subject; ii) treating said subject with an immunostimulatory drug when the expressions of NKp30c and NKp44c for said subject are the two highest among said group.

18. The method of claim 6, wherein the stem cells are allogeneic cardiac stem cells.

19. The method of claim 7, wherein the haematological malignancy is leukemia, lymphoma or multiple myeloma.

Description

FIGURE LEGENDS

[0062] FIG. 1. dNK cells and pNK cells differentially express NCRs isoforms.

[0063] mRNA were extracted from freshly-isolated dNK and pNK cells that were purified from the same donor. Relative expression of the three NKp30 (a, b, c) and NKp44 (d, e, f) splice variants as determined by quantitative reverse transcription PCR (qRT-PCR) analysis. (a, d) Relative mRNA expression. (b, e) mRNA ratios calculated for each cell type. (c, f) mRNA relative expression ratios between dNK and pNK cells. Data are representative of ten independent donors. Bar graphs are means.e.m. *P<0.05 and **P<0.01, ns: not significant.

[0064] FIG. 2. dNK and pNK cells are differentially activated after NCR cross-linking.

[0065] Freshly-isolated dNK and IL15-pNK cells were stimulated for four hours (a) with a single or a combination of two specific mAb, used as ligands. NK cell degranulation was assessed by quantification of CD107a cell surface expression using flow cytometry on CD3.sup.negCD56.sup.pos cells. (b) Percentage of freshly-isolated dNK cells and (c) pNK cells showing CD107a surface expression. Results presented as mean valuess.e.m. from 4 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

[0066] FIG. 3. Decidual cytokine environment impacts pNK cell NCRs isoform expression.

[0067] Comparative expression of (a) NKp30a, (b) NKp30b, and (c) NKp30c mRNA isoforms in pNK cells cultured in media supplemented with IL15, IL18 and TGF-, alone or in combination, relative to expression in pNK cells cultured in complete media. Bar graphs represented mean valuess.e.m. from four independent experiments. (d) Comparative expression of NKp44a, (e) NKp44b, and (f) NKp44c mRNA isoforms in pNK cells cultured in media supplemented with IL15, IL18 and TGF- alone or in combination, relative to expression in pNK cells cultured in complete media. Bar graphs represented means.e.m. from four independent experiments. (g) Fold induction of NKp30 and (h) NKp44 mRNA isoforms in six days cultured pNK cells relative to expression in pNK cells cultured in complete media. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

[0068] FIG. 4. NCR expression is modulated by pregnancy cytokines cocktail.

[0069] Mean Fluorescence Intensity (MFI) from four independent donors after six days culture with cytokine cocktail. Graphs represent freshly isolated dNK cells, pNK cells cultured in complete media or pNK cells in media supplemented with cytokine cocktail. Data on the graphs represent mean values.e.m. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

[0070] FIG. 5. Decidual cytokine environment impacts pNK cell function.

[0071] (a) CD107a cell surface expression on 6-days cultured pNK cells and freshly isolated dNK cells after 4 hours of NCR-ligation. NK cells were stimulated with anti-NKp30, -NKp44, -NKp46 antibodies or Isotype matched controls. Cell degranulation (CD107a expression) was assessed by flow cytometry on CD3.sup.negCD56.sup.pos cells. Representative graphs of 6 independent experiments are presented. (b) Secreted TNF- (c), IFN- (d), and VEGF-A (e) by 6-days cultured pNK cells and freshly isolated dNK cells was measured by multiplexed assay after 18 hours NKp30-, NKp44- or NKp46-ligation. Supernatants were collected from 3 independent experiments. Values represent means.e.m. of triplicates within the same experiment. *P<0.05, **P<0.01, ***P<0.001.

EXAMPLE 1: NKP30/NCR3 AND NKP44/NCR2 MICROENVIRONMENT-INURED ALTERNATIVE SPLICED VARIANTS DELINEATE DISTINCT NK CELL SUBSETS ORCHESTRATING THEIR FUNCTION

Materials and Methods

Ethics Statement

[0072] Informed consents were signed before samples were taken (Agence de la Biomdecine, PFS08-022, France).

[0073] Cell Purification

[0074] First-trimester decidua basalis (8-12 weeks of pregnancy) were obtained after elective termination of pregnancy as previously describes.sup.13. decidua basalis samples were minced and collagenase IV treated (Sigma-Aldrich, France). dNK cells were purified from non-adherent cell fraction using MACS negative selection kits (Miltenyi Biotec, France). pNK cells were isolated from healthy blood donors and stimulated or not with 10 ng/ml of IL15 overnight. More than 98% of purified cells are CD3.sup.negCD56.sup.pos.

qRT-PCR

[0075] Total cellular RNA was isolated from dNK or pNK cells using RNeasy kit (Qiagen, France). First-strand cDNA was synthesized from 1 g of total RNA using SuperScript III reverse transcriptase and random primers according to manufacturer's procedures (Life Technologies, France). PCR primers for NCR3.sup.9, NCR2 and the -actin housekeeping transcripts were designed using NCBI primer blast (Table 1 below).

TABLE-US-00001 TABLE1 Sequencesofforwardandreverseprimersusedfor qRT-PCRanalyses.Primersweredesignedusing NCBIBlastandpurchasedfromSigma(Sigma, France). Name Sequence actin Forward 5-CAAACATGATCTGGGTCATCTTCTC-3 (SEQIDNO:1) actin Reverse 5-GCTCGTCGTTCGACAACGGCT-3 (SEQIDNO:2) NKp30/NCR3 Forward 5-TTTCCTCCATGACCACCAGG-3 (SEQIDNO:3) NKp30/NCR3 Reverse 5-TTCTTGGACCTTTCCAGG-3 (a) (SEQIDNO:4) NKp30/NCR3 Reverse 5-CGGAGAGAGTAGATTTGGCATATT-3 (b) (SEQIDNO:5) NKp30/NCR3 Reverse 5-TTCCCATGTGACAGTGGCATT-3 (c) (SEQIDNO:6) NKp44/NCR2 Forward 5-AAGCCCCTGAGTCTCCATCT-3 (a) (SEQIDNO:7) NKp44/NCR2 Reverse 5-GTTTTCCACCATATGTCCCCC-3 (a) (SEQIDNO:8) NKp44/NCR2 Forward 5-TTCACAGACCCAGACCCAGAG-3 (b) (SEQIDNO:9) NKp44/NCR2 Reverse 5-AGGACGGGTGTGAAGGGACA-3 (b) (SEQIDNO:10) NKp44/NCR2 Forward 5-GTCCCTTCACAGCCACAGAA-3 (c) (SEQIDNO:11) NKp44/NCR2 Reverse 5-GAGACCTCCCTTGATGCTGC-3 (c) (SEQIDNO:12)

[0076] The qRT-PCR data were determined and normalized to the actin housekeeping gene according to the standard 2.sup.Ct method.

Immunoblotting

[0077] NK cells were stimulated for 20 min through receptor cross-linking on anti-NKp30-(clone-210847), anti-NKp44- (polyclonal goat IgG) or anti-NKp46-specific antibodies (clone-195314) coated plates. Cells were lysed in sample buffer (1% NP40, 20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1 mM PMSF, 1% glycerol and cocktail of proteases and phosphatases inhibitors). 8 g of proteins separated on 12% SDS-PAGE were electrotransferred to Immobilon membranes, immunoblotted with anti-phosphotyrosine antibody (4G10R-Platinum, Millipore) and normalized to -actin (MAB1501, clone-C4). Immunoreactivity was detected by chemiluminescence autoradiography (Sigma, Germany).

Degranulation Assay

[0078] For degranulation assay, NK cells were stimulated through receptor ligation using antibody-coated tissue culture plates (10 g/ml). Cells were stained with fluorochrome-conjugated anti-human CD107a (BD-Pharmingen) or isotype matched control then analyzed by Flow Cytometry. Histograms were obtained by applying a gate on CD3.sup.negCD56.sup.pos cells and analyzed using FlowJo software 7.6.5.

[0079] NK cells phenotype pNK cells were cultured in the presence of indicated cytokines (2.5 ng/ml of TGF- and 10 ng/ml of IL15 or IL18) for 6 days. Media were refreshed every 72 hours. Cells were immunostained with fluorochrome-conjugated antibodies: anti-CD56-APC, anti-CD3-PE-Cy7, anti-CD16-PE, anti-CD69-FITC, anti-NKG2D-PE, anti-NKG2A-PE, anti-NKp30-PE, anti-NKp44-PE, anti-NKp46-PE or anti-NKG2C-FITC (BD-Pharmingen). Histograms were obtained by applying a gate on CD3.sup.negCD56.sup.pos cells and analyzed using FlowJo software 7.6.5.

Confocal Microscopy

[0080] NK cells stimulated for 20 min on anti-NKp30, anti-NKp44, anti-NKp46 or anti-NKG2A coated glass-coverslips. Cells were paraformaldehyde fixed and stained with anti-perforin and anti-tubulin antibodies as previously described.sup.16. Filamentous actin cytoskeleton was visualized with AlexaFluor-conjugated phalloidin and nuclei stained with DAPI. Immune synapses (IS) were analyzed using Zeiss LSM710 confocal microscope (Carl Zeiss, Germany). Images were processed using ImageJ software.

Mutiplex Cytokine and Chemokine Array

[0081] pNK cells were cultured in the presence of indicated cytokine (2.5 ng/ml of TGF- and 10 ng/ml of IL15 or IL18) for 6 days. Cultured-pNK or freshly isolated dNK cells were stimulated through NCR-ligation for 18 hours. Cytokines, chemokines and growth factors levels were measured in culture supernatants by 7-multiplexed Affymetrix cytokine assay (TNF-, IFN-, VEGF-A, CXCL8/IL-8, CCL3/MIP-1, CCL4/MIP-1 and CXCL10/IP-10) according to the manufacturer's procedures (Procarta/eBioscience, France).

Statistical Analysis

[0082] Comparisons among independent groups were made using a 2-tailed Student t test. Data are expressed as mean valuestandard error of the mean (s.e.m.). P values less than 0.05 were considered significant. * P<0.05, ** P<0.01, *** P<0.001.

Results

[0083] dNK and pNK Cells Display Differential Expression of NKp30 and NKp44 Splice Variants

[0084] NCR-engagement triggers different effector functions in dNK and pNK cells.sup.13. Therefore, the inventors investigated whether alternatively spliced variants of NCR might individualize these two NK cells subsets in a cohort of dNK and pNK cells from the same donors.

[0085] Consistent with previous report.sup.9, pNK cells displayed predominant expression of NKp30a and NKp30b mRNA but almost no NKp30c mRNA (FIG. 1a). In contrast, dNK cells significantly expressed lower amounts of NKp30a (p=0.0047) and NKp30b (p=0.05) mRNA but very high levels of NKp30c mRNA (p=0.0034) (FIG. 1a). Differences between dNK cells and pNK cells were further highlighted by the relative mRNA expression ratio (FIG. 1b). dNK cells express 8- to 10-fold higher amounts of NKp30c, while they express NKp30a and NKp30b at significantly lower levels. Compared to pNK cells, dNK cells express at least 3.5-fold more NKp30c (FIG. 1c). On the other hand, dNK cells displayed similar expression levels of all three NKp44 mRNAs while pNK cells exclusively expressed NKp44b mRNA (FIG. 1d). Relative ratios further demonstrated that freshly isolated pNK cells express 12-fold more NKp44b than NKp44c mRNAs (FIG. 1e). dNK cells express at least 3- and 4-time higher quantities of NKp44a and NKp44c mRNAs than pNK cells (FIG. 1f). Thus, dNK cells predominantly express NKp30c, NKp44a and NKp44c whereas pNK cells mainly express NKp30a, NKp30b and NKp44b. This differential expression of NKp30 and NKp44 isoforms could individualize these two NK cells subsets at the molecular level.

[0086] Differential Expression of NCRs Splice Variants Impacts NK Cell Cytotoxicity

[0087] To examine the relevance of this dNK and pNK molecular individualization, the inventors investigated the impact of differential expression of NCR splice variants on cells effector functions. The inventors monitored cellular degranulation of dNK and pNK cells upon their NCR-ligation as readout for lytic activity. CD3.sup.negCD56.sup.pos pNK and dNK cells were activated for 4 hours with anti-NCR antibodies and analyzed for cell surface expression of CD107a degranulation marker (FIG. 2). Ligation of NKp30 receptor but not NKp44, IgG isotype matched control (FIG. 2) or NKG2A inhibitory receptor (data not shown) on dNK cells resulted in a small increase of CD107a expression (P=0.05), while ligation of NKp46 induced dNK cell degranulation (FIG. 2a,b). Co-engagement of NKp44 and NKp46 receptors resulted in more than 30% decrease in NKp46-induced degranulation in dNK cells (P=0.0012) while co-ligation of NKp30 had no effect (FIG. 2a,b). In line with previous reports.sup.23-25, NKp30-, NKp44-, or NKp46-ligation resulted in significant increase of CD107a expression on pNK cells (FIG. 2a,c). Simultaneous NKp44- and NKp46-ligation showed an incremental effect on the ability of pNK cells to degranulate while co-engagement of NKp30 and NKp46 had no impact on NKp46-induced degranulation (FIG. 2a,c).

[0088] Together, these data demonstrate that only NKp46-ligation triggers robust lytic granule exocytosis in dNK cells. While NKp30 prompted mainly by isoform c induces modest degranulation in these cells, NKp44 prompted mainly by isoforms a and c acts rather as an inhibitory receptor. In contrast, all three NCRs similarly favor pNK cell degranulation. Thus, the molecular individualization of dNK and pNK cells is biologically or functionally relevant.

[0089] NCRs Splice Variants Impact NK Cell Immune Synapse (IS) Formation

[0090] Cytotoxic activity of NK cells is a dynamic process orchestrated in different steps. Receptor ligation leads to recruitment and activation of signaling pathways. To validate the relevance of observed molecular individualization of dNK and pNK subsets, the inventors sought to provide mechanistic insights into the differential functions of NCR isoforms. First the inventors analyzed tyrosine phosphorylation after NCR-ligation. Upon NKp30- or NKp44-ligation tyrosine phosphorylation patterns were quite different between the two NK cell populations. However, phosphorylation patterns were similar after NKp46-ligation on dNK and pNK cells (data not shown). These differences suggest that the engagement of the same NCR would trigger the recruitment of different signaling pathways in dNK or pNK cells.

[0091] Immune synapse formation is associated with cortical actin remodeling. The organization of actin-containing micro-domains at the vicinity of the MTOC serves as a docking site for lytic granules.sup.26. To provide further mechanistic insights, the inventors then examined the impact of differential tyrosine phosphorylation patterns on the molecular organization of IS after single NCR-ligation on pNK and dNK cells (data not shown). NKp30-ligation resulted in a rapid reorganization of F-actin enriched cytoskeleton and MTOC and polarization of lytic granules in more than 45% of pNK cells (data not shown). By contrast, less than 20% of dNK cells showed polarized lytic granules after NKp30-ligation. Similar to NKp30, NKp44-ligation induced organized IS in more than 50% of pNK cells while only minor effects were seen in dNK cells (data not shown). Finally, activation through NKp46-ligation had similar effects on pNK or dNK cells with almost 50% of cells sharing features of cytolytic IS (data not shown). Similar to IgG control (data not shown), less than 20% of cells showed organized IS after ligation of control NKG2A inhibitory receptor (data not shown).

[0092] Overall, these data demonstrate that recruitment of different signaling pathways by NKp30 and NKp44 in dNK and pNK cells significantly impacts IS formation and cytotoxic activity of these two NK cells subsets. In line with the cytotoxic activity, all three NCRs favor pNK cell lytic IS formation while only NKp46-ligation triggers lytic IS in dNK cells. Thus, differential NCR splice variants expression by dNK and pNK is biologically and functionally relevant, which could probably contribute to promote their distinct functioning.

[0093] Decidual Microenvironment Modulates the mRNA Expression of pNK Cell NCR Isoforms

[0094] The above findings promoted us to investigate the elements that might contribute to sculpturing the expression of NCRs splice variants in NK cells subsets. dNK and pNK cells operate within distinct microenvironment. dNK operate within maternal endometrium enriched with immunomodulatory proteins (TGF-) and pro-inflammatory cytokines such as IL15 and IL18 that are produced by the decidual stroma hosting fetal trophoblast.sup.27-29. Therefore, the inventors analyzed the potential role of these cytokines in leading the expression of NCR splice variants in pNK toward the expression profile of dNK cells. pNK cells were cultured in the presence of TGF-/IL15/IL18 cocktail as well as other cytokine combinations as indicated (FIG. 3) and their mRNAs levels of NKp30 and NKp44 transcripts were evaluated. Compared to untreated-pNK cells, IL15 induced significant increase of NKp30a and NKp30b mRNA (P=0.0035 and 0.0142 respectively). Increases were also observed for NKp30c mRNA but did not reach significant levels due to variations amongst individuals. Treatment with TGF- induced significant decrease in the relative mRNA level of all three transcripts (FIG. 3a,b,c). The presence of IL15 tempers the TGF- effect although this does not reach significance for NKp30b and NKp30c splice variants. IL18 alone significantly increases the basic level of NKp30a splice variant mRNA (P=0.029) but its presence does not override TGF- effect (FIG. 3a,b,c). Finally, the TGF-/IL15/IL18 cocktail induces significant increases of the basic level of all three NKp30 transcripts (P=0.0254, 0.009 and 0.0325 respectively) (FIG. 3a,b,c). Moreover, when compared to each other, increases of NKp30b (4.5-fold) and NKp30c (4.3-fold) induced by the TGF-/IL15/IL18 cocktail dominate that of NKp30a (2.9-fold, P=0.014) (FIG. 3g).

[0095] Next, the inventors examined the expression of NKp44 splice variants. The presence of IL15, IL18 or TGF- alone or in combination induces significant changes in the expression level of NKp44a and NKp44c without affecting the expression of NKp44b mRNA (FIG. 3d,e,f). IL15 increases the basal level of NKp44a, whereas IL18 rather down regulates NKp44a. IL15/IL18 in the presence or absence of TGF- induces the highest up-regulation of this mRNA (P=0.015) (FIG. 3d,e,f). IL18 is more effective in increasing the basic level of NKp44c isoform (P=0.0006) and again IL15/IL18 in the presence (P=0.0018) or absence (P=0.0346) of TGF- significantly up-regulates its expression (FIG. 3f). Compared to NKp44b, TGF-/IL15/IL18 induces seven-fold increase of NKp44a (14-fold, P=0.0195) and significantly higher increase of NKp44c (P=0.0005) (FIG. 3h).

[0096] Thus, a microenvironment rich in TGF-/IL15/IL18 combination shifts the expression of NKp30 and NKp44 splice variants in pNK cells toward that of dNK cells. The data attribute an important role for cytokinic microenvironment in sculpturing and maintaining molecular individualization of NK cells subsets at least in terms of NKp30 and NKp44 splice variants mRNA transcription in pNK and dNK cells.

[0097] Decidual Microenvironment Impacts pNK Cell Phenotype

[0098] To support that microenvironment-inured NKp30/NCR3 and NKp44/NCR2 alternative spliced variants polarize NK cells subsets, the inventors analyzed the potential role of TGF-/IL15/IL18 cocktail and other cytokine combinations in modulating NK cell phenotype.

[0099] In the presence of TGF-/IL15/IL18 and consistent with previous reports.sup.30, pNK cells up-regulated their CD56 expression and more than 98% of the cells become CD56.sup.bright. Cells showed significant increases of Mean Fluorescence Intensity (MFI) 200.69.6 instead of 20.72.6 for cells cultured in medium (P<0.0001), reaching levels observed for dNK cells (2598.4) (FIG. 4). Lower CD56 MFI increases were also observed when pNK cells were cultured with IL15 (11712, P=0.0129) or TGF-/IL15 (1644, P=0.001). The presence of IL15 also significantly increased CD69 expression, which is barely expressed on freshly isolated pNK cells (FIG. 4) (P=0.0002 for IL15 alone, P=0.006 for IL15/IL18, P=0.005 for TGF-/IL15 or P=0.001 for TGF-/IL15/IL18). More than 40% of cytokine-cultured pNK cells expressed CD69 with MFI of 38.45 (FIG. 4) reaching the level of dNK cells (455). Furthermore, the presence of IL15 allowed the maintenance of CD16 expression in more than 70% of cells whereas only minor changes were observed for other receptors including NKG2D, NKG2C and NKG2A (data not shown) controlling the specificity of observed results.

[0100] The inventors next analyzed NCRs expression on pNK cells. More than 60% of pNK cells express NKp30. Treatment with TGF-/IL15/IL18 does not affect the expression level of NKp30, which is decreased under other culture conditions (FIG. 4). Treatment of pNK cells with IL15 alone or in combination with the other two cytokines induced NKp44 expression in 60% of cells (data not shown). MFI comparison showed very low variability amongst different donors, with TGF-13/IL15/IL18 treatment shifting pNK cells towards dNK cell phenotype (FIG. 4). Finally, the presence of IL15 was able to up-regulate NKp46 MFI but addition of TGF- had no effect. These changes were not due to cell proliferation or cell death. Only slight increases of proliferation were observed after six days of culture with IL15 and cell death was negligible.

[0101] Thus, similar to their effect on expression of NKp30 and NKp44 splice variants, TGF-/IL15/IL18 cytokines sculpt the phenotype of pNK cells shifting it towards dNK cells phenotype. This finding supports that the phenotype and probably the effector functions of various NK cell subsets, namely dNK and pNK cells in this report, are commanded at least in part by their cytokine microenvironment.

[0102] Decidual Microenvironment Impacts pNK Cell Function

[0103] The inventors then tested whether microenvironment-inured NKp30/NCR3 and NKp44/NCR2 alternative changes in pNK cell phenotype would impact their effector functions. Cells were cultured in different cytokine combinations and their lytic function was assessed through analyses of CD107a expression after NCR-ligation by flow cytometry (FIG. 5a). While pNK cells cultured in medium show low capacity to degranulate, IL15-maintained pNK cells significantly degranulate upon NKp30-(427.6%), NKp46- (24.75.9%) and, to a much lesser extent, NKp44-ligation (8.10.5%). IL15/IL18 combination showed similar results to IL15 alone. Treatment with TGF- did not affect basal level of CD107a expression in pNK cells. However, TGF-/IL15 or TGF-/IL15/IL18 significantly decreased the capacity of NKp30- and to lesser extent NKp44-stimulation to induce CD107a expression (17.8% and 5.7% respectively) but it did not blunt the response through NKp46 (FIG. 5a). These data indicate that pNK cells have normal capacities to degranulate upon NCR engagement and TGF- does not affect the NCRs response in the same manner.

[0104] The inventors next compared cytokine secretion by pNK cells, maintained under different conditions, to that of dNK cells after NCR-ligation (FIG. 5). Cells were stimulated through NCR-ligation and supernatants were analyzed by multiplexed assay (TNF-, IFN-, VEGF-A, CCL3, CCL4, CXCL8 and CXCL10). Medium-maintained pNK cells showed very low capacity to produce TNF-, IFN- or VEGF-A but they produced substantial amounts CCL4 in response to NKp30- or NKp46-ligation. Only minor changes were observed for CCL3, CXCL8 and CXCL10. The presence of IL15 induced strong increases of TNF-, IFN-, VEGF-A, CCL3, CCL4 and CXCL8 after NCR-ligation but did not affect the basal level of CXCL10 secretion (FIG. 5). IL18 alone had only minor effect on cytokine production and IL15/IL18 showed capacity similar to IL15 alone. TGF- alone showed minor effects. Compared to IL15, TGF-/IL15 or TGF-/IL15/IL18 treatment strongly decreased the capacity of NKp30-ligation to induce high amount of TNF-, IFN- and CCL3 (FIG. 5b,c) but increased VEGF-A production (FIG. 5d). Similar to NKp30, NKp44-ligation showed decrease in TNF-, IFN-, CCL3 and CCL4 but did not affect the secretion of VEGF-A (FIG. 5). The addition of TGF- did not impair NKp46-induced secretion of TNF-, IFN- or CCL3 of IL15-cultured pNK cells (FIG. 5). Compared to IL15-treatment NKp46-ligation resulted in increased VEGF-A production in TGF-/IL15- or TGF-/IL15/IL18-treated cells (FIG. 5d).

[0105] Taken together, these data suggest that the combination of TGF-, IL15 and IL18, results in pNK cells behaving similar to dNK cells since they secrete large amounts of VEGF-A and very low amounts of IFN-, TNF- in response to NKp30- or NKp44-ligation. However, only minor changes are observed upon NKp46-ligation in TGF-/IL15/IL18-treated cells.

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