Killer Ig-like receptor
Natural cytotoxicity receptor
The cognate NK–DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A+KIR– NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.
NK cells are effector members of the innate immune system that play a major role in host defense against tumor cells and pathogen-infected cells 1, 2. Theirfunction is regulated by a balance between activating and inhibitory signals that are generated by an array of different cell-surface receptors upon engagement by specific cellular ligands. Among the various inhibitory receptors, of primary importance are those specific for MHC class I molecules. These receptors, by delivering inhibitory signals to NK cells, can prevent unwanted responses to normal cells that express a complete set of self-MHC molecules 3–5. Remarkably, to escape CTL-mediated immune surveillance, certain tumor cells and virus-infectedcells have evolved mechanisms responsible for down-regulation of MHC class I expression 6, 7. This, however, results in the lack of engagement of the inhibitory receptors of NK cells, thus leading to lysis of these abnormal target cells. The activating receptors mediating the NK cell triggering against (HLA-deficient) abnormal cells are represented by a heterogeneousfamily of NK-cell-specific Ig-like molecules that have been termed "natural cytotoxicity receptors" (NCR; these include NKp30, NKp44 and NKp46) 8. Another triggering-receptor functionally related to NCR is NKG2D. Whereas the cellular ligands recognized by NKG2D have recently been identified, those recognized by NCR are still undefined 9, 10].
The balance between signals provided by MHC class I-specific inhibitory receptors and by the various triggering receptors is the basis of the ability of NK cells to discriminate between normaland abnormal autologous cells. A remarkable exception is represented by the capability of NK cells to kill normal autologous DC. In this context, it has recently been shown that the cognate interaction between NK cells and DC results in a potent cross-signaling 11–16. Thus DC can prime and induce proliferation of NK cells, which, in turn, once activated, acquire the ability to kill immature DC (iDC) and may also induce DC maturation. The NK-cell-mediated killing of iDC in peripheral tissues can be interpreted as a negative regulation of DC function, allowing the innate immune system to select the most appropriate DC that undergo maturation after Ag uptake in inflamed tissues 14. It has been reported that mature DC (mDC), unlike iDC, are generally resistant to NK-cell-mediated cytotoxicity. The different susceptibility of iDC versus mDC to NK-cell-mediated killing is thought to be mostly based on the differential expression of HLA class I molecules. Thus, although both iDC and mDC express HLA class I, the surface density of these molecules is greatly increased in mDC 17.
In the present study we investigated whether heterogeneity existed among NK cells in their capability to kill monocyte-derived DC. To this end, we analyzed NK clones expressing different inhibitory receptors for HLA class I molecules. We show that killing of iDC is not a function common to all NK cells but is rather a property of an NK cell population characterized by the CD94/ NKG2A+KIR– surface phenotype. Accordingly, the susceptibility of iDC to NK-cell-mediated killing reflects the sharp down-regulation of HLA-E (i.e. the cellular ligand for CD94/NKG2A) in iDC. Finally, we show that a small subset of CD94/NKG2A+KIR– NK cells is capable of also killing mDC. The NK cell functional capability analyzed in this study may be crucial to allow a potential, NK-dependent, "editing" process 14 by which the most appropriate mDC are selected for subsequent priming of naive cells in secondary lymphoid compartments.
2.1 Killing of autologous iDC is mediated by a subset of CD94/NKG2A+KIR– NK cells
Polyclonal NK cells cultured in the presence of exogenous IL-2 were previously shown to display strong cytolytic activity against iDC 11, 13, 18–21. Accordingly, in the present study, polyclonal NK cell populations isolated from donors AM, AC and DB efficiently killed both autologous and allogeneic iDC. However, the cytolytic activity against autologous iDC could be incremented in the presence of appropriate anti-HLA class I mAb (Fig. 1A). These data could be the consequence of the disruption of inhibitory interactions occurring between self HLA class I on DC and inhibitory receptors on NK cells.
On the basis of these results, we formulated the hypothesis that only a fraction of the total NK cell pool displays spontaneous cytotoxicity against iDC whereas the other NK cells do not because of effective inhibitory interactions between their receptors and HLA class I molecules. To analyze this possibility, a panel of NK cell clones isolated from donors AM, AC and DB were assessed for cytolytic activity against autologous (and allogeneic) iDC. Consistent with our hypothesis, only a fraction of NK cell clones lysed autologous iDC (Fig. 1B). The other clones displayed either little or no cytotoxicity. Moreover, the percentage of cytolytic clones was slightly increased when target cells were represented by allogeneic iDC (see below).
To verify whether the inability of certain NK cell clones to lyse iDC reflected the interaction of their inhibitory NKR with HLA class I molecules, these clones were analyzed for the ability to lyse autologous iDC either in the absence or in the presence of anti-HLA class I mAb (i.e. under conditions that disrupt the inhibitory interactions). On the basis of the results of these experiments, NK cell clones were grouped into three different functional categories and further analyzed for the expression of HLA class I-specific inhibitory receptors including killer Ig-like receptor (KIR)2DL, KIR3DL1 and CD94/NKG2A (i.e. the main MHC class I-specific inhibitory receptors in humans).
The first group (group A) of NK clones was characterized by high spontaneous cytolytic activity against iDC. The magnitude of their cytolytic activity could not, or could only minimally, be increased in the presence of anti-HLA class I mAb. These clones were rather homogeneous in terms of expression of inhibitory receptors as they expressed CD94/NKG2A but lacked KIR2DL and KIR3DL1, which react with self-HLA class I alleles (see two representative clones AM17 and DB33 in Fig. 2A).
The second group of NK cell clones (group B) was also characterized by the capability of spontaneously killing iDC. However, at variance with group A clones, their cytotoxicity increased in the presence of anti-HLA class I mAb. This suggested the occurrence of inhibitory interactions that limited, but did not abrogate, the NK-cell-mediated cytolysis. This group was also composed of CD94/NKG2A+ clones and lacked KIR reactive with self-HLA class I alleles. Remarkably, the cytolytic activity of group B NK clones could also be incremented in the presence of anti-CD94 mAb thus indicating that the (partial) inhibition of cytotoxicity was indeed mediated by CD94/NKG2A (see representative clones AM36 and DB1 in Fig. 2A).
NK clones belonging to the third group (group C) did not display cytotoxicity against autologous iDC. However, in the presence of anti-HLA class I mAb, iDC were efficiently lysed, suggesting the occurrence of potent inhibitory interactions. These NK clones were more heterogeneous regarding the expression of inhibitory receptors. Remarkably, virtually all NK clones expressing KIR2DL or KIR3DL1 specific for self-HLA class I alleles were included in this group (see clones DB3 and AM10 in Fig. 2). Moreover, some of these clones were characterized by the expression of a single KIR whereas others expressed multiple KIR with different specificities 3–6. The reconstitution of cytolytic activity against iDC could be obtained not only with anti-HLA class I mAb but also with anti-KIR mAb (see below). Finally a minor fraction of group C NK cell clones was KIR–CD94/NKG2A+ (see clone AM2 in Fig. 2A). Their cytotoxicity could be reconstituted by mAb-mediated blocking of CD94 or by anti-HLA class I mAb.
These data indicate that: (a) Not all NK cells are capable of killing autologous iDC (although all NK cells could lyse iDC in the presence of anti-HLA class I mAb); (b) clones displaying spontaneous cytolytic activity against iDC are restricted to an NK subset characterized by the CD94/ NKG2A+KIR– surface phenotype (groups A and B); (c) clones expressing KIR2DL or KIR3DL1, which are specific for self-HLA class I alleles, do not kill autologous iDC (group C).
Some NK clones expressed both self-reactive KIR and CD94/NKG2A. In all instances, they were confined to group C and their cytolytic activity could be reconstituted both by anti-HLA class I and anti-KIR mAb, whereas anti-CD94 mAb had little or no effect (see the representative clone AM10 in Fig. 2A). Finally it is worth mentioning that KIR+NKG2A– clones were found to display cytolytic activity against iDC only in experiments in which iDC were derived from allogeneic (KIR-mismatched) individuals 22. In this case, KIR+NKG2A– cells display alloreactivity because the expressed KIR fail to recognize HLA class I alleles on allogeneic DC. As shown in Fig. 2B, the representative NK clone AM4 (KIR3DL1+) was unable to kill autologous iDC (BW4+BW6–) whereas it lysed allogeneic, KIR-mismatched (BW4–BW6+) iDC. Killing of autologous iDC could be reconstituted in the presence of anti-HLA class I mAb whereas killing of allogeneic iDC was not significantly modified. Another example indicating the ability of KIR to distinguish between autologous and allogeneic, KIR-mismatched, iDC is provided by clone DB3, which co-expresses KIR2DL1 and KIR2DL2. This clone can be defined as "non-alloreactive" because, on the basis of its KIR phenotype, should recognize all different HLA-C alleles (both group 1 and group 2) 23. Indeed this clone did not kill autologous or allogeneic iDC whereas lysis of both targets could be efficiently reconstituted by anti-HLA class I mAb. Moreover, reconstitution of lysis was obtained by anti-KIR2DL2 mAb against autologous (CW1/CW3) iDC and by anti-KIR2DL1 mAb against allogeneic (CW2/CW4) iDC (Fig. 2B) Finally, as expected, in the case of NKG2A+KIR– clones (see clone AM36 in Fig. 2B) no substantial difference existed in the ability to kill autologous or allogeneic iDC.
2.2 The susceptibility of iDC to NK-mediated cytotoxicity reflects the down-modulation of HLA-E class I molecules
Previous studies demonstrated that iDC and mDC display remarkable differences in terms of HLA class I surface expression 17. Thus, by the use of mAb specific for a monomorphic determinant of HLA-A, B, C and E molecules, it has been shown that DC undergoing maturation greatly up-regulate their HLA class I expression at the cell surface. Moreover, the up-regulation of HLA class I represented a crucial mechanism by which mDC become resistant to NK-cell-mediated lysis.
To directly assess the expression of various HLA class I molecules on cells representative of different stages of DC maturation we comparatively analyzed the expression of HLA-A, B, C and E on monocytes, iDC and mDC derived from the same individual. As shown in Fig. 3, all HLA class I molecules were highly up-regulated in mDC as compared with iDC. Remarkably, they were clearly down-regulated in iDC as compared with monocytes (i.e. the precursors of iDC). Thus, it appears that the generation of iDC from monocytes results not only in the acquisition (or up-regulation) of novel surface molecules (for example CD1a) and functional properties 24–27 but also in the loss (or down-regulation) of the expression of various molecules 24 including CD14, and HLA-A, B, C and E molecules (this report). This would suggest that the degree of HLA class I down-regulation is tuned to levels that allow iDC to become sensitive to lysis mediated by a particular subset of NK cells (CD94/ NKG2A+KIR–).
Along this line, because KIR+ NK cells are unable to kill iDC, it is conceivable that the amount of HLA-B or HLA-C molecules expressed by iDC is sufficient to generate KIR cross-linking and delivery of inhibitory signals. On the other hand, the down-regulation of HLA-E would be sufficient to enable a fraction of KIR–NKG2A+ NK cells to kill iDC. Indeed it can be seen (Fig. 3) that HLA-E (as detected by the HLA-E-specific 3D12 mAb) 28 was almost undetectable in iDC whereas it was only partially re-expressed on mDC. However, in all instances, the HLA-E expression in mDC was lower as compared with monocytes (Fig. 3) or PBL (not shown) derived from the same individual. Surprisingly, although HLA-A, B and C molecules were expressed by mDC at levels higher than by PHA blasts, the surface expression of HLA-E was consistently lower in mDC than in PHA blasts. In this context, previous studies provided clear evidence that autologous PHA blasts are highly resistant to NK lysis independently of the KIR/NKG2A phenotype of the effector NK cells 3.
2.3 A small fraction of NK clones can mediate killing of mDC
Consistent with previous reports that polyclonal NK cells do not efficiently kill mDC 11, 13, 16, 21 we show that most NK cell clones that lysed iDC did not to kill mDC. Interestingly, however, mDC were lysed by a minor fraction of NK clones belonging to group A (i.e. those displaying spontaneous anti-iDC cytolytic activity that could not be increased by anti-HLA class I mAb) (Fig. 4). Lysis of autologous mDC was lower as compared with that of iDC and could be increased in the presence of anti-HLA class I mAb (for comparison, see Fig. 2A and Fig. 4). This suggests that the higher expression of HLA-E in mDC as compared with iDC results in a more effective signaling via CD94/NKG2A (this is also suggested by the ability of anti-CD94 mAb to increase their lysis). Concerning group B NK clones (i.e. capable of killing iDC and whose lysis was incremented by anti-HLA class I mAb), they displayed no cytolytic activity again mDC; however, cytolytic activity could be revealed in the presence of anti-HLA class I or anti-CD94 mAb (see clones AM36 and DB1 in Fig. 4). Finally clones belonging to group C (in most instances KIR+), which are unable to kill iDC, also failed to kill mDC. Cytotoxicity against mDC could only be detected upon mAb-mediated disruption of the interaction between HLA class I and KIR (see clones DB3 and AM10 in Fig. 4).
2.4 Heterogeneity of KIR–NKG2A+ NK cells in the ability to kill DC
As illustrated above, NK cell clones belonging to group A and B are characterized by a homogeneous KIR–NKG2A+ surface phenotype whereas group C includes either KIR+ NKG2A– or KIR– NKG2A+ clones, (or, less frequently, KIR+NKG2A+ clones). Assuming that the negative signaling via KIR is more effective than that via NKG2A, (either because of an intrinsic difference in their signaling capability or because of the different availability of the specific HLA class I ligands on DC) it should be clarified why KIR–NKG2A+ cells are detectable in all three groups of NK clones. Since the cytolytic activity of a given NK cell clone is the result of a balance between inhibitory (KIR, NKG2A) and triggering (NCR, NKG2D) receptors, we analyzed the levels of expression of these molecules in the different groups of NK clones. In particular, we focused our attention on the expression of NKG2A and of NKp30 (i.e. the triggering NCR that plays a predominant role in the induction of NK-cell-mediated lysis of iDC and mDC) 11.
First, the NKG2A+KIR– clones belonging to group A, B and C were evaluated for the level of NKG2A surface expression. As shown in Fig. 5, NK clones belonging to group C expressed very high levels of NKG2A as compared with groups A and B. Moreover, group A clones were characterized by a lower expression of NKG2A as compared with group B clones. These data suggest the existence of an inverse correlation between the levels of NKG2A expression and the ability to kill iDC (and mDC). The low amounts of HLA-E molecules expressed in iDC may be differentially sensed by NK cells expressing high or low levels of NKG2A whereas mDC (expressing higher levels of HLA-E) are susceptible to lysis only by NK clones characterized by very low NKG2A surface density.
Regarding the expression of NKp30, this was comparable in most NKG2A+ clones analyzed (Fig. 5). Consistent with these data, their ability to kill iDC in the presence of anti-HLA class I mAb (i.e. in the absence of inhibitory interactions) did not show significant differences (see Fig. 2 and Fig. 4).
In this study we show that human NK cells display major differences in their ability to kill autologous DC. Thus, the cytotoxicity against iDC is restricted to a subset of NKG2A+KIR– NK cells, whereas NK cells that express KIR specific for self HLA class I alleles do not kill. Heterogeneity exists even among NKG2A+KIR– cells in the magnitude of cytolytic responses. This appears to inversely correlate with the surface density of NKG2A. Accordingly, NK clones expressing low levels of NKG2A (group A) lysed both iDC and mDC whereas those expressing higher levels of NKG2A killed only iDC or, in a few cases, (NKG2Abright) failed to kill both iDC and mDC.
It has been suggested that, in inflamed tissues, the NK-cell-mediated cytotoxicity against iDC may serve to positively select those DC that upon Ag uptake undergo maturation 14, 15. The maturation process is characterized by profound changes in DC function. These include the down-regulation of the Ag capture capability, as well as changes in chemokine receptor expression and the acquisition of costimulatory molecules required for naive T cell priming 25–30. However, in the context of the NK–DC interaction, a most important event in the process of DC maturation is the up-regulation of the surface expression of HLA class I molecules. Along this line, our data suggest that the quality-control of DC undergoing maturation in peripheral tissues is mediated by NKG2A+KIR– NK cells that keep in check the expression of HLA-E at the DC surface. Notably, we also show that the surface expression of HLA-E is sharply reduced in iDC as compared with monocytes whereas it is partially recovered in mDC. On the contrary, the reduced cell surface levels of HLA-B and HLA-C in iDC are still sufficient to effectively engage KIR3DL1 or KIR2DL. Accordingly, KIR+ NK cell clones did not kill autologous iDC (while they could kill allogeneic, KIR-mismatched, iDC).
The finding that killing of autologous iDC is mediated by NKG2A+KIR– NK cells but not by KIR+ NK cells cannot be explained on the basis of differences in the expression of NKp30 (i.e. the receptor primarily involved in the NK-cell-mediated killing of DC) since lysis of autologous iDC by KIR+ NK cells could be restored in the presence of anti-HLA class I or anti-KIR mAb (Fig. 2A, B). Moreover, KIR+ clones efficiently lysed allogeneic iDC derived from "KIR-mismatched" individuals (Fig. 2B). Thus one may speculate that KIR+ NK cells are not involved in the selection of autologous iDC undergoing maturation in peripheral tissues and, more generally, that KIR+ NK cells may not participate in the NK/DC cross-talk 14, 15.
Importantly, as shown recently, NK cells undergo in vitro activation and proliferation upon interaction with autologous or allogeneic iDC 11–16. Along this line, it will be interesting to analyze whether both KIR+ and KIR– NK cells give rise to proliferative responses in the presence of iDC. It is conceivable that the interaction between KIR and HLA class I molecules might affect not only NK cell cytotoxicity but also their proliferative responses. If this holds true, one may predict that in autologous NK–DC interactions only a fraction of NK cells confined to the NKG2A+KIR– population would actually proliferate.
A different situation can be envisaged in the interaction between NK cells and allogeneic DC. In this case, KIR+ NK cells displaying a mismatch between KIR and HLA class I would undergo proliferation and kill both iDC and mDC. This possibility is consistent with the finding that, in haplotype-mismatched individuals undergoing BM transplantation, alloreactive NK cells, derived from hematopoietic cell precursors, can prevent T cell priming by killing allogeneic DC while sparing non-hematopoietic tissues 22.
An unexpected finding was the identification of a small subset of NK cell clones belonging to group A (5–10%) that were capable of killing autologous mDC. These NK clones do not express self-reactive KIR and are characterized by low levels of NKG2A (see Fig. 5). This allows these NK cells to readily sense the down-regulation of HLA-E on target cells as compared with NK cells expressing higher levels of NKG2A. Accordingly no increases of the cytolytic activity of NKG2Alow NK cells against iDC occurred in the presence of anti-HLA class I mAb. On the other hand, in the case of mDC (expressing higher levels of HLA-E), addition of anti-HLA class I mAb resulted in an increase of cytolytic activity, indicating that, provided a sufficient level of receptor–ligand interaction, NKG2A molecules expressed by group A clones can inhibit lysis. It is conceivable that in mDC some degree of heterogeneity might exist in the expression of HLA-E and, possibly, of ligand(s) of NKp30. Given the ability of a fraction of NK cells to discriminate between cells that express different amounts of HLA-E, it is possible that among mDC only some may express a surface density of HLA-E sufficient to confer resistance to this particular subset of NK cells.
What could be the physiological role of NK cells capable of killing autologous mDC? Both DC undergoing maturation (mDC) and a fraction of NK cells express CCR7, a chemokine receptor allowing migration into secondary lymphoid organs 14, 31–35. Thus, it is tempting to speculate that NK cells capable of killing mDC may be capable of migrating together with mDC. Once in secondary lymphoid tissues, these NK cells could contribute to the "editing" process of mDC and regulate the type of downstream interactions between mDC and T cells 14.
To validate this hypothesis it will be important to evaluate whether these NK cells, under appropriate conditions (perhaps during interaction with DC, or perhaps in response to certain cytokines or chemokines) may express CCR7. Along this line, CCR7+ cells have been identified within the CD56brightCD16– population in peripheral blood NK cells 2, 34, 35. In most instances this population is characterized by the CD94/NKG2A+KIR– phenotype. Although NK clones belonging to group A did not express surface CCR7 while they did express CD16 (not shown), it is conceivable that the microenvironmental conditions that induce CCR7 expression and down-regulation CD16 in vivo may be rather different from the in vitro conditions required for NK cell proliferation and cloning.
It has been shown that NK cells, in the presence of pro-inflammatory stimuli, can favor the progression of DC maturation. Apparently, this phenomenon involves TNF-α that is released by DC upon direct contact with NK cells and/or by NK cells themselves 13. It would be interesting to verify whether these "regulatory" NK cells may be identified by the analysis of NK cell clones carrying different NKG2A, KIR phenotypes.
In conclusion, our present data provide further information on the complex interactions occurring between NK cells and DC. They also offer a clear example of the functional heterogeneity of NKcells and how this may reflect on the control of DC survival and, consequently, on downstream functions, including the magnitude and the quality of T cell responses.
4 Materials and methods
The following mAb, produced in our laboratory, were used in this study: JT3A (IgG2a, anti-CD3), AZ20 and F252 (IgG1 and IgM, respectively, anti-NKp30), c127 (IgG1, anti-CD16), c218 (IgG1, anti-CD56), EB6b (IgG1, anti-KIR2DL1 and KIR2DS1), GL183 (IgG1, anti-KIR2DL2 KIR2DL3 and KIR2DS2), FES172 (IgG2a, anti-KIR2DS4), Z27 (IgG1, anti-KIR3DL1), XA185 (IgG1, anti-CD94), Z199 (IgG2b, anti-NKG2A), A6–136 (IgM, anti-HLA class I), 131 (IgG1, anti-HLA-A alleles including A3, A11 and A24) and E59/53 (IgG2a, anti-HLA-A) 36, 37. The mAb F4/326 (IgG, anti-HLA-C) 38, 116-5-28 (IgG2a, anti-HLA-Bw4 alleles) and 126–39 (IgG3, anti-HLA-Bw6 alleles) 37 were kindly provided by Dr K. Gelsthorpe (Sheffield, GB) (XII International HLA Workshop) and 3D12 (IgG1, anti-HLA-E) 28 was kindly provided by Dr. Daniel Geraghty (Fred Hutchinson Cancer Research Center, Seattle, WA).
Anti-CD1a (IgG1–PE), anti-CD14 (IgG2a), anti-CD83 (IgG2b) and anti-CD86 (IgG2b–PE) were purchased from Immunotech (Marseille, France).
D1.12 (IgG2a, anti-HLA-DR) mAb was provided by Dr R. S. Accolla (Pavia, Italy). HP2.6 (IgG2a, anti-CD4) mAb was provided by Dr P. Sanchez-Madrid (Madrid, Spain).
4.2 Generation of polyclonal or clonal NK cell populations
To obtain PBL, PBMC were isolated on Ficoll-Hypaque gradients and depleted of plastic-adherent cells. Enriched NK cells were isolated by incubating PBL with anti-CD3 (JT3A), anti-CD4 (HP2.6) and anti-HLA-DR (D1.12) mAb (30 min at 4°C) followed by goat anti-mouse coated Dynabeads (Dynal, Oslo, Norway) (30 min at 4°C) and immunomagnetic depletion. CD3–CD4–HLA-DR–cells were cultured on irradiated feeder cells in the presence of 100 U/ml rIL-2 (Proleukin, Chiron Corp., Emeryville, CA) and 1.5 ng/ml PHA (Gibco Ltd, Paisley, GB) to obtain polyclonal NK cell populations or, after limiting dilution, NK cell clones as previously described 23.
4.3 Generation of DC
DC were generated as previously described 17. Briefly, PBMC were derived from healthy donors and plastic-adherent cells were cultured in the presence of IL-4 and GM-CSF (Peprotech, London, GB) at a final concentration of 20 ng/ml and 50 ng/ml, respectively. After 6 days of culture, cells were characterized by the CD14–CD1a+CD83– phenotype corresponding to iDC. To generate CD14–CD1a +CD83+CD86+ mDC, iDC were stimulated for 2 days with LPS (Sigma-Aldrich, St. Louis, MI) at a final concentration of 1 μg/ml.
4.4 Flow cytofluorimetric analysis and cytolytic activity
For one- or two-color cytofluorimetric analysis (FACSCalibur, Becton Dickinson and Co., Mountain View, CA), cells were stained with the appropriate mAb followed by PE- or FITC-conjugated isotype-specific goat anti-mouse second reagent (Southern Biotechnology Associated, Birmingham, AL) as described previously 23. Polyclonal and clonal NK cell populations were tested for cytolytic activity in a 4-h [51Cr]-release assay against either autologous or heterologous DC 23. The concentrations of the various mAb added were 10 μg/ml for masking experiments. The E:T ratios are indicated in the text.
This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero della Sanità, Ministero dell'Università e della Ricerca Scientifica e Tecnologica (M.U.R.S.T.) and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. Also the financial support of Fondazione Compagnia di San Paolo, Torino, Italy, is gratefully acknowledged. We thank Ms Tiziana Baffi for secretarial assistance.