International Immunology Advance Access published online on October 25, 2007
International Immunology, doi:10.1093/intimm/dxm105
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Heterogeneity of TLR3 mRNA transcripts and responsiveness to poly (I:C) in human NK cells derived from different donors
1 Dipartimento di Medicina Sperimentale, Università di Genova, Genova, Italy
2 Istituto Giannina Gaslini, Genova, Italy
3 Centro di Eccellenza per la Ricerca Biomedica, Genova, Italy
Correspondence to: Correspondence to: A. Moretta; E-mail: alemoret{at}unige.it
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Abstract |
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TLR3 plays an important role in the activation of different cell types of the innate immune system. Previous studies indicated that human NK cells express TLR3 and that, upon stimulation by polyinosinic-polycytidylic acid [poly (I:C)], they release cytokines and up-regulate cytotoxicity. Here we show that NK cells display heterogeneous levels of TLR3 mRNA transcript. Analysis of NK cell clones did not reveal significant correlation between the levels of TLR3 mRNA transcripts and the expression of different surface NK receptors including killer Ig-like receptor and NKG2A. On the other hand, the level of TLR3 mRNA transcript detected in given clones correlated with the ability of these clones to respond to poly (I:C). Thus, clones displaying higher TLR3 mRNA transcripts were characterized by higher cytokine production and cytotoxicity. Moreover, the increased cytolytic activity induced by treatment with poly (I:C) does not depend on increment of the expression of activating NK receptors and co-receptors, adhesion molecules or perforin/granzyme, but correlates with higher cell responsiveness to NKp46 ligation. Remarkably, in the presence of poly (I:C), even NKp46dull NK cell clones become cytolytic when characterized by high levels of TLR3 transcript. Thus, our present study provides an useful tool for both a quantitative and qualitative analysis of TLR3 in NK cells and contributes to explain the heterogeneous responsiveness to poly (I:C) of NK cells derived from different individuals.
Keywords: cytokine release, innate immunity, NK-mediated cytotoxicity, Toll-like receptor
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Introduction |
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Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, and induce antimicrobial immune responses (1, 2). Human TLRs sense distinct microbial components conserved among pathogens but absent in humans, and elicit different, but sometimes overlapping, immune responses. Ten members of the TLR family have been identified in humans so far. Some TLRs, such as TLR3, TLR7 and TLR8, seem to be sensors of viral infections. In particular, TLR3 recognizes double-stranded RNA (dsRNA) of viral origin (3, 4), whereas TLR7 (5–7) and TLR8 (5, 7) recognize single-stranded RNA (ssRNA). All these TLRs together with TLR9, that recognizes unmethylated deoxycytidyl-deoxyguanosine (CpG) motifs characteristic of microbial DNA (8–10), sense nucleoside-based ligands and are required for effective anti-viral defense. Remarkably, TLRs responsible for viral recognition (TLR3, TLR7, TLR8 and TLR9) are expressed in intracellular compartments (6, 11–15). Moreover, recently two cytoplasmic proteins, able to detect dsRNA, have been identified: retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (mda-5) (16–18). RIG-I and mda-5 are structurally related and are characterized by a helicase domain, that is involved in dsRNA recognition, and two caspase-recruiting domain-like domains that are responsible for the activation of the downstream signaling.
We have previously shown that treatment of freshly isolated peripheral blood NK cells with stimuli acting on TLR3 leads to surface expression of CD69 and CD25 activation markers, IFN-
and tumor necrosis factor (TNF)-
production and up-regulation of cytolytic activity against tumor cell lines or monocyte-derived immature dendritic cell (iDC). The capability of responding to TLR3 was not confined to NK cells freshly isolated from peripheral blood, but it was also a property of activated NK cells (19).
In this study, we analyzed a panel of human NK cell clones derived from three different donors for the expression of TLR3 mRNA and for their ability to respond to polyinosinic-polycytidylic acid [poly (I:C)] stimulation.
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Methods |
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Monoclonal antibodies
The following mAbs were used in this study: JT3A (IgG2a anti-CD3), c127 (IgG1 anti-CD16), c218 (IgG1 anti-CD56), BAB281 and KL247 (IgG1 and IgM, respectively, anti-NKp46), AZ20 and F252 (IgG1 and IgM, respectively, anti-NKp30), Z231 and KS38 (IgG1 and IgM, respectively, anti-NKp44), ON72 (IgG1 anti-NKG2D), XA185 (IgG1 anti-CD94), Z270 (IgG1 anti-NKG2A), EB6 (IgG1 anti-KIR2DL1/S1), GL183 (IgG1 anti-KIR2DL2/L3/S2), FS172 (IgG2a anti-KIR2DS4), Z27 (IgG1 anti-KIR3DL1/S1), Q66 (IgM anti-KIR3DL2), PP35 (IgG1 anti-2B4), ON56 (IgG2b anti-NTB-A), MAR206 (IgG1 anti-CD2) and c284 (IgG1 anti-CD18) were produced in our laboratory.
D1.12 (IgG2a anti-HLA-DR) and HP2.6 (IgG2a anti-CD4) mAb were kindly provided by R. S.Accolla (Università di Pavia, Pavia, Italy) and P. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain), respectively.
Perforin and granzyme B expression analysis in NK cells were performed using purified anti-perforin mAb (Ancell Corp., Bayport, MN, USA) and purified anti-granzyme B mAb (Alexis Biochemicals Corp., San Diego, CA, USA), respectively, after cells were fixed in 3% PFA and permeabilized.
Generation of NK cell clones
PBMC were derived from healthy donors by Ficoll-Hypaque (Biochrom AG, Berlin, Germany) gradients. PBMC were depleted of plastic-adherent cells and incubated with anti-CD3, anti-CD4 and anti-HLA-DR mAb (30 min at 4°C), followed by goat anti-mouse-coated Dynabeads (Dynal, Oslo, Norway) (30 min at 4°C) and immunomagnetic depletion. After limiting dilution CD3–4–DR– cells were cultured on irradiated feeder cells in the presence of 100 U ml–1 rhIL-2 (Proleukin; Chiron Corp., Emeryville, CA, USA) and 1.5 ng/ml PHA (Gibco Ltd, Paisley, Scotland) to obtain NK cell clones as previously described (20).
Analysis of TLR3, RIG-I and mda-5 transcripts in NK cell clones
Total RNA was extracted from NK cell clones and NK92 cell line using RNeasy mini kit (Qiagen) according to the manufacturer's instruction and cDNA synthesis was performed on 500 ng of RNA using hexameric primers. To exclude that PCR amplifications were due to DNA contaminations, RNA was treated with RNase-free DNase (Qiagen) and further amplified with or without retrotranscription. PCR amplifications were performed using TaqMan assay (Applied Biosystem 7700 Sequence Detector, Foster City, CA, USA). A two-step PCR procedure of 15 s at 95°C and 1 min at 60°C was applied for 40 cycles. The primers used for TLR3-specific amplification were Q-TLR3 up: 3' CCT GGT TTG TTA ATT GGA TTA ACG A 5' and Q-TLR3 down: 3' TGA GGT GGA GTG TTG CAA AGG 5', whereas the TLR3 probe was 3' ACC CAT ACC AAC ATC CCT GAG CTG TCA A 5'-6-FAM (21). The Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to normalize the TLR3 quantity (human GAPDH Endogenous control kit; Applied Biosystem). The normalized TLR3 mRNA transcript of the tested samples was calculated as time-fold mRNA detected in the NK cell line NK92 (chosen as reference in this study). Each clone was analyzed in two independent experiments and each reaction was performed at least in triplicate.
Four different sets of primers were used for end-point PCR analysis. The primer sequences used for TLR3 amplification (1360 bp) were TLR3 up: 5' CAA GCA GAA GAA TTT AAT CAC and TLR3 down: 5' TTA TTC AAT CCT AAA TCG ATG, those utilized for RIG-I analysis were RIG-I up: 5' TGT GTG CTT CTC TTG ATG and RIG-I down: 5' TTC CTG TGT TCT GAT TTG, while those used for mda-5 transcript detection were mda-5 up: 5' AGC TGC AAA AAA AGG AAA T and mda-5 down: 5' GTG ATG CAT TTT CTC AAT T. Finally, the set of primers b act- up: 5' ACT CCA TCA TGA AGT GTG ACG and b act-down: 5' CAT ACT CCT GCT TGC TGA TCC allowed to amplify a 250-bp fragment of human beta actin cDNA. The amplification profiles were 30 cycles: 30 s at 94°C, 30 s at 58°C, 30 s at 72°C for mda-5 and b act PCRs, while RIG-I and TLR3 amplifications required 33 cycles PCRs and an annealing temperature of 58 and of 53°C, respectively. The PCR products were resolved into a 1% agarose gel.
TLR-mediated stimulation of NK cell clones
NK cell clones derived from three different donors were cultured for 20 h in 24-well plates at a concentration of 1 x 106 in 1 ml of RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, 1% penicillin-streptomycin-neomycin and 20 U ml–1 rhIL-2 (Proleukin; Chiron Corp.) in the absence or in the presence of 25 µg ml–1 poly (I:C) (Amersham Biotech, Buckinghamshire, UK).
Cytokine analysis
Supernatants collected from unstimulated and poly (I:C)-stimulated NK cell clones were analyzed for the IFN- and TNF- content using ELISA kits from Biosource International, Inc. (Camarillo, CA, USA) following the manufacturer's instructions.
Phenotypic analysis of NK cell clones
Cells were incubated with appropriate mAb followed by PE- or FITC-conjugated isotype-specific goat anti-mouse secondary reagent (Southern Biotechnology Associated, Birmingham, AL, USA). Samples were analyzed by one- or two-color cytofluorimetric analysis (FACScan; Becton Dickinson & Co., Mountain View, CA, USA) as previously described (20).
Cell lines and cytolytic activity of NK cell clones
The cell lines used as targets in the various cytolytic assays were the following: 221 (LCL721.221, human EBV-transformed B cell line), M14 (human melanoma), P815 (murine mastocytoma) and FO-1 (human melanoma). The NK-mediated cytotoxicity was assessed in a 4-h [51Cr] release assay as previously described. For the masking experiments, the concentration of KL247 mAb was 10 µg ml–1. The effector/target (E/T) ratios are indicated in the text.
Statistical analysis
For inter-group comparisons, Mann–Whitney test was used. All calculations were performed using the Prism software package (release 3.00; GraphPad Software Inc., San Diego, CA, USA).
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Results |
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Human NK cell clones display heterogeneous levels of TLR3 mRNA transcripts
In a previous study, we observed that the ability of NK cells to respond to poly (I:C) may greatly differ in different donors. We also showed that two distinct NK cell clones displayed major differences in their responses to TLR3 and TLR9 stimulation (19).
In the present study, a large panel of NK cell clones derived from three different donors was assessed for the levels of expression of TLR3. Recently, commercial anti-TLR3 mAb has been shown to recognize also an epitope expressed by a putative centrosomal protein (12). Thus, we decided to avoid the cytofluorimetric approach for the detection of intra-cytoplasmic TLR3 in NK cell clones and we chose to assess the expression of TLR3 by quantitative TaqMan analysis. Thus, 75 unstimulated NK cell clones derived from three donors (donor CRI n = 35, donor DP n = 25 and donor ZO n = 15) were selected and analyzed. The NK92 cell line was used as reference and the amount of TLR3 mRNA transcript detected in NK cell clones was reported as time-fold the TLR3 mRNA expressed in this NK cell line. The results of these experiments are summarized in Fig. 1. It is evident that a remarkable heterogeneity exists in terms of TLR3 transcript among the NK cell clones analyzed. Moreover, different mean values for TLR3 expression existed in NK clones from different donors (i.e. CRI 
= 1.78, DP 
, and ZO 
time-fold NK92 TLR3 transcript). According to the levels of TLR3 mRNA, three groups of NK cell clones could be identified in each donor. Thus, for example, in donor CRI, 9 NK cell clones were characterized by low levels of expression (<0.5 time-fold NK92 transcript) (group 1), 9 expressed levels of TLR3 mRNA transcripts ranging from 0.5 to 1.5 time-fold (group 2), while 17 clones displayed high levels of expression (>1.5 time-fold) (group 3). In donor DP, 14 NK cell clones belonged to group 1, 6 NK cell clones to group 2 and 5 NK cell clones to group 3. In donor ZO, nine NK cell clones belonged to group 1, five NK cell clones to group 2 and one NK cell clone to group 3.
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These data suggest that heterogeneity in TLR3 mRNA transcript exists among different NK cell clones even when derived from the same donor. Moreover, they also suggest that the proportions of NK cell clones characterized by high levels of TLR3 transcript may differ among different donors. This finding may provide a rationale for the heterogeneity of poly (I:C) responsiveness observed among different donors.
We further investigated whether NK cell clones, belonging to the above-mentioned groups, were characterized by a particular surface phenotype. To this end, we assessed the surface expression of different receptors including natural cytotoxicity receptors (NCRs) (NKp46, NKp30 and NKp44), killer Ig-like receptors (KIRs), NKG2A and CD16. As shown in Fig. 2, the various NK cell clones were grouped on the basis on their reactivity (or lack thereof) with different anti-KIR mAbs including 11Pb6, GL183, Z27 and Q66 specific for KIR2DL1/S1, KIR2DL2/L3/S2, KIR3DL1/S1 and KIR3DL2, respectively. The various groups were then compared for their mean TLR3 transcript content. It can be seen that no significant differences could be detected between a given KIR phenotype and TLR3 expression with the exception of a weak correlation between the expression of KIR3DL1/S1 and a higher content of TLR3 mRNA (P = 0.0263).
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No correlation could be found also in the case of activating receptors since the levels of NCR and CD16 surface expression did not significantly match with those of TLR3 mRNA transcripts.
Correlation between levels of TLR3 mRNA transcript and responsiveness to poly (I:C) stimulation in NK cell clones
We next analyzed whether NK cell clones characterized by different amounts of TLR3 mRNA transcripts would display different capabilities of responding to stimuli acting on TLR3. To this end, NK cell clones representative of the three groups identified above, were first cultured for 2 days with low concentration of IL-2 and then assessed for their capability of responding to poly (I:C). The NK cell clones used in these studies were expanded for several weeks in the presence of exogenous r-IL-2 but in the absence of feeder cells. Thus, the observed effects of poly (I:C) are consequent to its direct interaction with NK cells and not with ‘contaminating’ feeder cells. As shown in Fig. 3, NK cell clones belonging to different groups released remarkably different levels of cytokines upon stimulation. Thus, treatment with poly (I:C) did not induce detectable IFN- or TNF- release by NK cell clones of group 1 (clones 5P6 and CRI215), whereas low to high cytokine levels were released by group 2 and group 3, respectively. The six NK cell clones shown in Fig. 3 were derived from two of the donors analyzed (donor DP and donor CRI) and are representative of the three groups of clones selected on the basis of the content of TLR3 transcript.
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These clones also displayed different levels of anti-tumor cytotoxicity measured after stimulation with poly (I:C). The tumor target cells used were represented by P815 (murine mastocytoma), M14 (human melanoma) and FO-1 (human melanoma). While P815 and M14 are known to be relatively resistant to NK cell lysis, FO-1 is highly susceptible.
As shown in Fig. 4, in poly (I:C)-treated group 1 NK cell clones, no up-regulation of cytolytic activity against the three target cells was detected. Treatment of group 2 clones induced low increments of cytolytic activity against P815 and M14, whereas treatment of group 3 clones resulted in a sharp increase of cytotoxicity against all three targets. Thus, in most NK cell clones, a correlation could be found between the degree of poly (I:C)-induced up-regulation of anti-tumor cytotoxicity and the content of TLR3 mRNA.
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At this regard, it is important to underline that the surface levels of adhesion molecules (including CD2 and LFA-1) and of activating receptors and co-receptors (i.e. NKp46, NKp30, NKp44, NKG2D, DNAM-1, 2B4 and NTBA) on NK cells did not increase after poly (I:C) treatment (data not shown).
It is also important to note that TLR3 does not represent the only innate sensor for dsRNA; indeed, two cytoplasmic proteins, named RIG-I and mda-5, have been recently described that may display similar function (2, 16–18). These two proteins are two helicases able to deliver an activating signal upon binding of viral dsRNA in the cytoplasm. Thus, we analyzed the expression of RIG-I and mda-5 transcripts by reverse transcription–PCR on total RNA isolated from human NK cell clones representative of the three groups identified above. As expected, the intensity of TLR3 end-point PCR products reflects the results obtained by quantitative PCR analysis (Fig. 5). Notably, our results suggested that RIG-I transcript was homogeneously expressed in all NK cell clones analyzed, whereas the levels of mda-5 transcript were heterogeneous among NK cell clones. Remarkably, however, the heterogeneity of mda-5 transcript did not correlate with responsiveness to poly (I:C).
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Taken together, these data suggest that the differential functional responsiveness to poly (I:C) of human NK cell clones correlates with the levels of TLR3 transcript but not with the levels of RIG-I and mda-5 transcript.
Poly (I:C) treatment increases NK cytotoxicity against tumor target cells that are recognized via the NKp46-activating receptor
Previous studies indicated that NKp46 plays a key role in the recognition and killing of certain tumor cell lines including P815 and M14 (22, 23). Thus, based on the above results, it was possible that the increase in NK cytolytic activity induced by poly (I:C) treatment could reflect the up-regulation of NKp46 surface expression or function. Since cytofluorimetric analysis did not reveal substantial increments in the levels of NKp46 expression upon poly (I:C) stimulation (data not shown), we investigated whether the increment of NK cytotoxicity induced by poly (I:C) treatment could depend on NKp46 functional up-regulation. To this end, we assessed the effect on cytotoxicity of a blocking anti-NKp46 mAb of IgM isotype. These experiments were carried out on a group of NK cell clones characterized by similar levels of TLR3 transcript but different levels of NKp46 surface expression. Thus, some clones were characterized by an NKp46bright phenotype (which is associated to higher anti-tumor cytolytic activity) while others by an NKp46dull phenotype (usually corresponding to clones displaying medium/low cytotoxicity) (20). Two representative NK cell clones (9P2 and 6P8) belonging to the above-mentioned group 3 were selected. As shown in Fig. 6(A), killing of M14 mediated by both NKp46bright (9P2) and NKp46dull (6P8) NK cell clones was sharply incremented upon poly (I:C) treatment. Anti-NKp46 mAb inhibited not only killing of M14 and P815 by unstimulated NK cell clones but also that of poly (I:C)-stimulated ones (Fig. 6A). Similar results were obtained also using LCL721.221 cell line (another target mainly killed via NKp46) (Fig. 6A).
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The functional up-regulation of the NK-mediated killing in response to poly (I:C) treatment is particularly striking in NKp46dull NK cell clones. As shown in Fig. 6(A), killing of M14, 221 and P815 by the unstimulated 6P8 clone was very low. This is in line with the low expression of NKp46. Remarkably, the cytolytic activity of this clone was strongly up-regulated by the treatment with poly (I:C) while the surface expression of NKp46 remained unchanged upon TLR3 engagement (Fig. 6B). Nevertheless, lysis of the three target cells could be strongly inhibited by mAb-mediated masking of NKp46 molecules. Thus, since anti-NKp46 mAb abrogates the killing by poly (I:C)-stimulated NK cells, it is conceivable that the observed up-regulation of cytotoxicity may be the result of an augmentation of the NKp46 triggering capability without involvement of additional de novo-expressed receptors.
Finally, it is important to note that the increment of NK cytotoxicity upon TLR3 engagement seems not to depend on increased content of perforin or granzymes into cytolytic granules. Indeed, the amounts of these cytoplasmic proteins in NK cell clones did not display significant variations after stimulation with poly (I:C) (data not shown).
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Discussion |
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NK cells have an important role in the early innate immunity against viral infections due to their rapid cytolytic activity and cytokine production. In inflamed tissues, NK cells can directly kill infected or transformed cells and rapidly secrete a variety of cytokines. This allows further recruitment and activation of other effector cell populations and contributes to the instructive phase of the adaptive immune response (24, 25). TLR3, that recognizes viral dsRNA, could play a central role in the host response to viruses.
In addition to the TLR3 response pathway, a TLR3-independent pathway able to recognize dsRNA has been recently described (2, 16–18). In particular, dsRNA phagocytosed from the extracellular space, where it is released by virally infected cells that undergo lysis or necrosis, is detected by TLR3 within endosomes, whereas dsRNA present in the cytosol of infected cells can be sensed by two different helicases termed RIG-I and mda-5.
Recently, NK cells have been described to express functional TLR3 (19, 26, 27). In particular, it was shown that both resting and activated NK cells are able to respond to poly (I:C) (19), a synthetic analog of viral dsRNA which has been used extensively in experimental studies to mimic viral infection. Moreover, it has been suggested the existence of a remarkable cross-talk between NK- and monocyte-derived dendritic cell (DC), possibly serving as a control switch between innate and adaptive immune response (24, 28). In particular, experimental evidence has been provided that poly (I:C) known to strongly activate monocyte-derived iDC can also act on NK cells. This effect is probably allowed by the release of cytokines (primarily IL-12) by DC during the early events of an inflammatory response. Thus, dsRNA of viral origin can simultaneously act on TLR3 expressed both by monocyte-derived iDC and NK cells recruited by chemokine gradients to the inflammatory sites. In the presence of IL-12, released by DC after antigen uptake, NK cells upon TLR3 engagement increase their anti-tumor cytotoxicity and acquire the capability of killing monocyte-derived iDC (19). Thus, NK cells can select the most appropriate DCs for subsequent T cell priming within secondary lymphoid compartments (19).
In this study, a large number of human NK cell clones were assessed for their level of TLR3 mRNA by TaqMan analysis. These experiments showed the existence of differences in the quantity of TLR3 mRNA transcript among NK cell clones derived from the same donor. Moreover, we could also detect a heterogeneous expression of TLR3 mRNA transcript among the three donors analyzed. Importantly, we demonstrate a correlation between the amount of TLR3 mRNA transcript, expressed in the NK cell clones, and their capability to respond to poly (I:C) stimulation. On the contrary, we could not detect a correlation between functional responsiveness to poly (I:C) and levels of expression of RIG-I and mda-5 transcripts. Notably, the proportion of NK cell clones expressing intermediate or high levels of TLR3 transcript was different in the three donors analyzed. These findings could explain the previous observation that NK cell populations, derived from different donors, can respond to TLR3 stimulation with different efficiency (19).
The different responsiveness of NK cells to poly (I:C) was established primarily by the assessment of cytokine production, but it was also confirmed by cytotoxicity assays against various target cell lines. In this regard, it is of note that cytokine release depends on the direct engagement of TLR3 on NK cell clones, whereas the increment of cytotoxicity also depends on the levels of expression of activating NK receptors and coreceptors, of adhesion molecules, as well as the perforin and granzyme content.
The use of different tumor target cells and of different effectors (NKp46bright or NKp46dull NK cell clones (20)) allowed the identification of the activating receptors that are functionally relevant in this phenomenon. Importantly, the poly (I:C)-induced up-regulation of NK-mediated killing was essentially evident only against a limited number of target cells. These targets are recognized primarily via NKp46. Indeed, both in the absence or in the presence of TLR3 stimulation, killing of P815, M14 and 221 target cells was strongly inhibited by anti-NKp46 mAbs (22, 23). Considering that poly (I:C) treatment did not result in an increment of expression of activating NK receptors and coreceptors, adhesion molecules or perforin/granzyme, it is possible that the up-regulation of cytotoxicity could depend on the activation of some tyrosine kinases involved in the signaling cascade associated with the NKp46 receptor. In this context, it is important to note that Pisegna et al. (26) have shown that the activity of p38 MAPK is involved in the up-regulation of cytolytic activity induced in NK cells by stimulation with dsRNA.
In most donors, mature NK cells express homogeneous levels of activating receptors and coreceptors; however, a small percentage (<10%) of otherwise normal individuals are characterized by a fraction of NK cells expressing an NCRdull phenotype. This phenotype results in defective ability to kill certain target cells that are primarily lysed via NCRs (20). In the present study, we have shown that NKp46dull NK cell clones, characterized by poor cytolytic activity, up-regulate their cytolytic activity upon treatment with poly (I:C), only when they express high levels of TLR3 mRNA transcript. In this context, it is important to stress that the surface levels of NKp46 did not increase after TLR3 stimulation. Thus, in the presence of dsRNA also, NK cell clones displaying an NKp46dull phenotype, but expressing high level of TLR3 transcript, up-regulate their function.
Several studies suggested that down-regulation of NCR expression may represent a mechanism by which tumor or virus-infected cells become resistant to NK-mediated lysis. For example, the NCRdull phenotype has been observed in over 90% of patients affected by acute myeloid leukemia (29), in HIV+ viremic patients (30), or upon NK cell exposure to corticosteroids in vitro as well as in patients treated with these drugs (31). Moreover, a strong inhibition of the surface expression of NKp30 and, in part, of NKG2D has been detected in vitro upon exposure of NK cells to TGFß (32). Thus, it could be interesting to analyze the effect of poly (I:C) stimulation also in these conditions to verify whether also these NCRdull NK cells may up-regulate their cytolytic activity.
In conclusion, our study provides evidence that TLR3 mRNA quantification represents a valuable tool to analyze the ability of different NK cell clones (and possibly of other cell types) to respond via TLR3. More importantly, based on the observed correlation between levels of TLR3 mRNA and ability to respond to poly (I:C) stimulation, our data suggest that the existence of heterogeneity among different donors can be explained by the different frequencies of NK cells expressing high levels of mRNA coding for this TLR.
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Funding |
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Associazione Italiana per la Ricerca sul Cancro; Istituto Superiore di Sanità; Ministero della Sanità; Ministero dell'Istruzione dell'Università e della Ricerca; Ministero della Salute–RF 2002/149; FIRB-MIUR progetto-codRBNE017B4; European Union FP6, LSHB-CT-2004-503319-AlloStem (the European Commission is not liable for any use that may be made of the information contained).
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Abbreviations |
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| DC, dendritic cell |
| dsRNA, double-stranded RNA |
| iDC, immature dendritic cell |
| KIR, killer Ig-like receptor |
| mda-5, melanoma differentiation-associated gene-5 |
| NCR, natural cytotoxicity receptor |
| poly (I:C), polyinosinic-polycytidylic acid |
| RIG-I, retinoic acid-inducible gene-I |
| ssRNA, single-stranded RNA |
| TLR, Toll-like receptor |
| TNF, tumor necrosis factor |
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Notes |
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* These authors contributed equally to this study.
Transmitting editor: E. Vivier
Received 27 July 2007, accepted 27 September 2007.
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References |
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