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Published online before print September 7, 2006
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* Laboratoire d’Immunogénétique-Allergologie, Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg-City, Luxembourg; and
Service de Médecine Interne, Clinique Médicale B, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
1Correspondence: Laboratoire d’Immunogénétique-Allergologie, Centre de Recherche Public de la Santé (CRP-Santé), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. E-mail: jacques.zimmer{at}crp-sante.lu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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Key Words: suppression • inhibition • TGF-ß • NKG2D
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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chain of the IL-2 receptor, they constitutively express CTLA-4, glucocorticoid-induced TNF receptor (GITR), and the transcription factor Foxp3 [1
2
3
]. These cells are crucial for the maintenance of peripheral tolerance and prevention of autoimmunity [1
2
3
4
]. They consequently inhibit proliferation and effector functions of a large panel of different cell types of the immune system (Fig. 1
): conventional CD4+ and CD8+ T lymphocytes [2
, 3
, 5
], NKT cells [6
], B cells [7
], dendritic cells (DC) [8
], and monocytes/macrophages [9
]. Besides TH1 and TH2 cells, IL-17-producing TH17 cells have been described recently as a third subset of CD4+ TH cells [10
, 11
]. These TH17 cells currently receive a lot of attention as a result of their importance in the induction of inflammation and autoimmunity [12
, 13
]. It remains, however, to be investigated if they are also targets of Treg-mediated suppression [13
]. Findings in this field would be likely to have significant implications for physiopathology and therapeutics of human inflammatory states.
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With the emphasis put on CD4+CD25+ Treg, it was simply a question of time until the appearance of papers describing their effects on NK cells, which are the third type of lymphocytes besides T and B cells. As an important component of the innate immune system, they have two major, functional properties (Fig. 2a
). The first one is cytotoxicity [18
, 19
], which can be subdivided further in natural cytotoxic activity, predominantly toward tumor cells and virally infected cells in the absence of prior stimulation or immunization, and ADCC, directed against antibody-coated target cells, the Fc part of the antibody triggering in that case the NK cell Fc
R CD16. Although present in resting NK cells, cytotoxicity increases strongly when NK cells are stimulated by cytokines such as IL-2, IL-15, IL-18, and many others. NK cell-mediated lysis of target cells is mediated mainly by the release of the cytotoxic molecules perforin and granzymes. The second important function of NK cells is cytokine production [18
, 19
], which occurs upon triggering of activating NK cell receptors and/or stimulation by cytokines present in the microenvironment. In particular, NK cells are an important source of IFN-. Cytotoxicity and cytokine production by NK cells contribute to innate immune responses and thus, to the first line of defense of the organism before adaptive immunity has developed. However, NK cells also participate directly in the induction and regulation of adaptive immune responses: they stimulate maturation of DC [20
, 21
], eliminate immature DC [20
, 21
], produce cytokines that influence CD4+ helper and CD8+ effector T cells [22
, 23
], and can regulate T cell activation and proliferation through direct cellular contacts [24
, 25
].
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–Rae1
, mouse ULBP-like transcript 1 (Mult1), and finally, H60, a minor histocompatibility antigen. One group [32
] identified an additional human ligand of NKG2D: lymphocyte effector cell toxicity-AL (Letal). These molecules are mostly absent from healthy cells but are induced upon cellular stress, as in the case of infection or malignant transformation [29
]. Ligands of the NCR (only NKp46 is present in human and mouse) are unknown presently [31
]. Viral proteins such as hemagglutinins [33
, 34
] and pp65 from human cytomegalovirus [35
] have been suggested but cannot be the only ligands, as NCR are implicated strongly in the killing of numerous tumor cell lines of different histologies [30
, 31
]. Furthermore, a ligand of NKp30 also seems to be expressed on human DC, as NK cell-mediated killing of immature DC depends on NKp30 engagement [36
]. A healthy cell expresses normal amounts of MHC Class I molecules and no or few ligands for major AR. Thus, upon encounter with a NK cell, an excess of inhibitory messages is transmitted to the NK cell via its IR, and the healthy cell is spared (Fig. 2b) . However, if the target cell becomes infected or malignant, it may lose expression of MHC Class I molecules partially (weak inhibition) or totally (no inhibition), whereas at the same time, stress-induced ligands for AR are up-regulated (strong activation). The balance in this case is clearly in favor of activation, so that the target cell will be lysed by the NK cell (Fig. 2b) [26 , 27 ].
Although this brief, general overview might, to some extent, introduce the topic, it cannot replace the sum of information contained in the excellent reviews that have been published recently about Treg [1 2 3 4 5 , 14 ] and NK cells [18 19 20 21 22 , 26 27 28 29 30 31 ], respectively (this list is not intended to be exhaustive).
As CD4+CD25+ Treg inhibit every cell type investigated so far, it was rather expected that they would also suppress NK cell functions, and this has indeed been confirmed independently by several groups in papers, which are with one exception, very recent [37 38 39 40 41 42 43 ]. The results are summarized in Figure 3 and in Table 1 . To our knowledge, only one short review about Treg-mediated inhibition of NK cells has been released previously [44 ], but it deals exclusively with the results obtained by Ghiringhelli et al. [37 ].
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CD4+CD25+ TREG INHIBIT NK CELLS IN VITRO |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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Comparable experiments were performed by Trzonkowski et al. [40
], although the authors used an unconventional way for cell activation: Purified human NK cells were cultured for 40 h together with autologous CD4+CD25+ or CD4+CD25– T cells and with autologous monocytes preincubated with anti-influenza vaccine to mimic in vivo stimulation by APC. When cytotoxic activity toward K562 targets was measured, it was significantly lower in Treg-containing cultures than in samples with CD4+CD25– T cells. In addition, there were fewer IFN-+ and perforin+ NK cells in the presence of Treg than in their absence. In a more recent follow-up study [45
] based on an identical methodology, the same group confirmed the previous results and moreover, detected an inverse correlation between Treg numbers in peripheral blood and level of cytotoxic activity of autologous PBMC.
In the study by Romagnani et al. [41 ], the interactions between human NK cells and plasmacytoid DC were investigated. When stimulated through TLR9, these DC stimulate NK cell activation (as assessed by the up-regulation of the activation marker CD69), cytotoxicity, and selectively in the CD56bright NK cell subset, proliferation. The presence of autologous CD4+CD25– T lymphocytes enhanced NK cell proliferation in an IL-2-dependent manner, whereas CD4+CD25high Treg completely abolished the enhancement. However, Treg had no effect on proliferation nor on up-regulation of CD69 induced in NK cells by plasmacytoid DC alone.
Smyth et al. [38
] used the C57BL/6 (B6) mouse model for their in vitro studies. Two murine tumor cell lines transfected with NKG2D ligands, the lymphoma RMA-S-Rae1ß and the melanoma B16-Rae1, were used as targets in cytotoxicity assays. As effectors, purified, splenic NK cells were added alone or together with CD4+CD25+ Treg or CD4+CD25– control T cells. NK cells alone as well as NK cells cocultured with conventional T cells efficiently killed both targets. In contrast, cytotoxicity was reduced significantly, although far from being abolished when Treg were present in the assay together with NK cells. Percentages of lysis were in that case like those observed with the parental lines RMA-S and B16, killed independently of NKG2D. These targets were lysed to the same extent in the presence or absence of Treg, at lower percentages than their Rae-transfected counterparts.
Also in a mouse system designed to study the role of Treg in chemically induced tumor development, Nishikawa et al. [42 ] confirmed that activated CD4+CD25+ Treg significantly inhibit cytotoxic activity of resting NK cells toward YAC-1 lymphoma targets. For that, they used 18 h cytotoxicity assays, which rather explore TNF superfamily-induced, late apoptotic cell death [46 ] instead of the conventional tests performed over 4 h, evaluating perforin- and granzyme-mediated rapid target cell lysis [46 ].
From a historical point of view, the first investigation of the phenomenon of Treg-mediated NK cell inhibition is probably the one by Shimizu et al. [43 ], published in 1999. They observed that when splenocytes from Balb/c, B6, or C3H mouse strains were depleted of CD4+CD25+ cells and afterwards cultured for 1 week in the absence of any stimulation, they efficiently lysed a panel of syngeneic and allogeneic tumor cell lines. Various control experiments demonstrated that the cytotoxic cells generated were most likely NK cells. However, when total Balb/c splenocytes (not depleted of Treg) were tested under the same conditions, no cytotoxic activity developed.
A potential point of concern with most of these in vitro experiments might be the possibility of some kind of cold target inhibition [47 ]. Indeed, the presence of an additional population of T lymphocytes besides effector NK cells and target tumor cells in the culture system might lead to conjugate formation of at least part of the NK cells with T cells, so that fewer NK cells would be available for target cell killing. It is interesting that Trzonkowski et al. [40 ] showed that NK cells selectively form conglomerates with CD4+CD25+ Treg but not with other purified lymphocyte subpopulations. Thus, inhibition of NK cell-mediated lysis of target cells could reflect the engagement of a significant fraction of the NK cells in conglomerates with Treg, the residual, low percentage of specific lysis being attributable to those rare NK cells that are not bound in the conglomerates. In this situation, Treg would not inhibit NK cell cytotoxicity directly but simply impede NK cell contact with target cells. This, however, does not seem to be the case, as the number of NK cell-target cell conjugates is not reduced in the presence of Treg compared with other lymphocytes [40 ]. Nevertheless, the interesting question of why NK cells form such conglomerates, specifically with CD4+CD25+ Treg but apparently not with other cell types contained in PBMC, remains to be resolved.
It is surprising that human Treg displayed a suppressive activity on NK cells without prior activation through their TCR in the study of Ghiringhelli et al. [37 ]. It is commonly accepted that such a TCR-mediated activation of CD4+CD25+ Treg is required in vitro to enable them to exert their regulatory functions [14 , 48 ]. In human, this has, for example, been demonstrated by Dieckmann et al. [49 ], who also showed that syngeneic DC alone were, in contrast to allogeneic DC, not sufficient for Treg activation, and by Jonuleit et al. [50 ]. As inhibition of NK cell-mediated cytotoxicity was also observed when Treg were first fixed with formaldehyde [37 ], which suggests that Treg membrane-bound molecules are involved in the inhibition, it might well be that Treg use different mechanisms to inhibit cytotoxicity of NK cells and proliferation of conventional T cells, with a need for prior activation in the latter case but not in the former. Although totally identical experiments were not performed with mouse Treg, these cells were activated systematically in vitro prior to adoptive transfer to recipient mice [37 ]. Likewise, Smyth et al. [38 ] showed that only activated but not unstimulated mouse Treg could inhibit NK cell-mediated cytotoxicity in vitro. These results strongly suggest the existence of a functional difference between human and mouse CD4+CD25+ Treg in this regard.
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CD4+CD25+ TREG INHIBIT NK CELLS IN VIVO |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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To further demonstrate that the absence of Treg might lead to NK cell stimulation, the authors assessed in vivo NK cell proliferation in Treg-deficient, Scurfy mice compared with wild-type mice and found that it was increased hugely in the former. Scurfy mice lack a functional gene coding for Foxp3, the master transcription factor for Treg development and function and thus, have no CD4+CD25+ Treg [1 , 3 , 4 ]. Not only proliferation but also natural cytotoxicity against YAC-1 was considerably higher in Scurfy than in wild-type mice. In normal animals, depletion of Treg by anti-CD25 antibody enhanced NK cell proliferation compared with control antibody-treated mice. Thus, these experiments suggest that Treg control and inhibit NK cells, also under normal, physiological conditions and not only in the context of tumor cell lysis.
Yet another argument for in vivo suppression of NK cells by Treg was provided through i.p. injection of MHC Class I-deficient RMA-S cells, which usually induces local recruitment and proliferation of NK cells [51 ]. This process was affected significantly when Treg but not conventional T lymphocytes were coinjected [37 ].
Smyth et al. [38 ] tested their hypothesis of Treg-mediated NK cell inhibition directly by depleting Treg (i.p. injection of an anti-CD25 antibody) from B6 mice before i.v. inoculation of the lung carcinoma cell line 3LL. Treg-depleted mice developed significantly fewer lung metastases than animals treated with a control antibody. That indeed NK cells, but not T cells, were responsible for the relative tumor resistance observed in Treg-depleted mice was demonstrated by the fact that the simultaneous removal of NK cells was followed by the development of a high number of lung metastases. It should however be kept in mind that the NK cell-depleting antibodies antiasialoGM1 and NK1.1 used in this study are also likely to eliminate some CD8+ T cells and NKT cells, respectively.
Host defense was abolished completely, if Treg were depleted or not, when a high dose of 3LL cells, which completely overwhelmed the immune response, was incoculated. Similar results were obtained with B16-Rae1 melanoma cells. A third tumor cell line, RMA-S-Rae1ß, was s.c.-injected, and primary tumor cell growth was monitored. In control antibody-injected mice, tumor size increased rapidly over a period of 30 days. In contrast, in Treg-depleted mice, tumors were controlled efficiently or even rejected completely, when tumor-free mice were kept under observation for at least 100 days [38
].
Comparable results were obtained through in vivo tumor metastasis suppression experiments: T cell-deficient RAG-1–/– mice were i.v.-injected with B16 or B16-Rae1 tumor cells and 6 h later, with activated Treg or CD4+CD25– T lymphocytes. Lung metastases, counted on Day 14, were most abundant in mice inoculated with B16-Rae1 and then with activated Treg. Equally high numbers of metastases observed in NK-depleted animals indicated that tumor rejection was indeed performed by NK cells.
The paper by Barao et al. [39 ] uses an alternative approach by investigating BMG rejection, which is a completely different topic from that of tumor cell lysis. In lethally irradiated rodent recipients, NK cells are responsible for the rejection of allogeneic and parental BMG. The latter situation is known as "hybrid resistance" [52 ]: NK cells from F1 hybrid mice reject BMG from either parent, as parent cells express only half of the MHC Class I molecules present in the recipient and are thus eliminated by these NK cells because of their "missing self."
First, Treg-depleted B6 (MHC haplotype: H2b) and (B6xBalb/c) F1 hybrid (MHC haplotype: H2bxd) mice were lethally irradiated and subsequently received Balb/c (MHC haplotype: H2d) BMG at cell doses in which resistance was only partial. The level of early bone marrow engraftment was assessed 6 days later by determining numbers of CFU-GM in the spleen of the recipients. High CFU-GM numbers reflect better engraftment, and low values reflect stronger rejection. In the fully allogeneic and hybrid-resistance situations, graft rejection was increased significantly in the Treg-depleted recipients compared with those that had received the nondepleting control antibody. Rejection was dependent on host NK cells, as it was impaired severely in NK cell-depleted recipients. It is clear from these experiments that NK cell-mediated rejection of allogeneic and parental BMG is strengthened significantly if Treg are removed from the recipient, and thus, the authors [39 ] provide yet another example showing that the Treg control NK cell functions in vivo.
Long-term survival and donor chimerism of grafted recipients depleted of Treg were also analyzed [39 ]. B6 recipients were treated with control or with anti-CD25 antibody, lethally irradiated, grafted with three different, increasing doses of Balb/c bone marrow cells, and assessed for survival and donor chimerism at Day 35. At lower doses, high percentages of Treg-depleted mice compared with control mice died as a result of bone marrow failure (likely reflecting complete NK cell-mediated rejection of the grafts). At higher doses, more or all mice survived, but the level of donor chimerism (percentage of H2Dd+ splenocytes) was higher in those recipients that had been treated with the control antibody than in those treated with anti-CD25 antibody. As a conclusion, one might consider that depletion of CD4+CD25+ Treg increases short-term BMG rejection and reduces long-term survival and donor chimerism.
Finally, the authors looked at the influence of adoptive transfer of CD4+CD25+ Treg on the rejection of H2b BMG by hybrid F1 H2bxd recipients. Sublethally irradiated F1 mice were grafted with T cell-depleted H2b bone marrow cells alone or together with purified H2b CD4+CD25+ Treg. One week later, the number of CFU-GM was increased significantly in mice reconstituted simultaneously with bone marrow cells and Treg compared with those treated with bone marrow alone. Thus, cotransferred Treg favor bone marrow engraftment and inhibit host NK cell-mediated graft rejection, a result that confirms the other data shown in this paper [39 ] by simply turning things the other way around.
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THE INHIBITION OF NK CELLS BY CD4+CD25+ TREG IS MEDIATED BY TGF-ß |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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In the model of Smyth et al. [38 ], TGF-ß also seems to play a major role: NK cell lysis of RMA-S-Rae1ß in the presence of Treg strongly increased when an anti-TGF-ß antibody but not an anti-IL-10 antibody was added. That TGF-ß indeed originated from Treg was shown by the absence of lysis increase when the anti-TGF-ß antibody was put in cytotoxicity assays performed with NK cells alone and NK cells mixed with conventional T cells. TGF-ß was membrane-bound rather than soluble, as inhibition did not occur when both cell types were separated by Transwells or when the supernatant of activated Treg cultures was used instead of Treg themselves.
Barao et al. [39 ] likewise demonstrate the involvement of TGF-ß in the inhibition of NK cell-mediated BMG rejection by Treg, as injection of a neutralizing anti-TGF-ß antibody, before allogeneic or parental BMG, increased rejection significantly. This means that NK cells were not inhibited in this situation.
Trzonkowski et al. [40 ] did not investigate TGF-ß production by Treg. Instead, they show that upon coculture with NK cells, 5–10% of Treg produced IL-10. Anti-IL-10 antibodies, however, did not influence suppressive actions on NK cells so that the immunosuppressive cytokine IL-10 does not seem to be at the origin of the inhibition.
It is interesting to note that the three papers [37 38 39 ], which thoroughly investigated the mechanism of Treg-mediated inhibition, all conclude independently that TGF-ß, most likely in its membrane-bound form [37 , 38 ], is the effector molecule responsible for NK cell inhibition. Indeed, there is, as already indicated above, a controversy in the literature about the actual role of TGF-ß as a mediator of immune suppression performed by CD4+CD25+ Treg, which is mentioned in most recent reviews about Treg biology [1 2 3 , 5 , 14 , 48 ] or TGF-ß [53 ]. Some groups describe the presence of membrane-bound TGF-ß on Treg [15 , 54 ] and claim it is responsible for their suppressive effects, whereas others do not confirm these findings [55 , 56 ], although TGF-ß is produced and released by activated human Treg [55 ]. Anti-TGF-ß antibodies do not interfere in vitro with Treg-mediated inhibition in a lot of studies [1 2 3 4 5 , 14 ], and moreover, Treg isolated from neonatal TGF-ß KO mice still suppress in vitro proliferation of normal CD4+ T cells efficiently [56 ]. Membrane-bound TGF-ß has also been reported to be down-regulated on human CD4+CD25+ Treg upon activation [48 ]. This might be one reason why Ghiringhelli et al. [37 ] used resting human Treg rather than activated ones. The usual, but possibly a bit too simple, explanation proposed for all these discrepancies is that different experimental protocols have been used [56 ]. As Treg-mediated inhibition is most frequently evaluated through the reduction of proliferation of conventional T cells, a way to reconcile some of the discordant findings could be to consider (and to prove experimentally) that TGF-ß is indeed not required for suppression of T cell proliferation but that in contrast, it is essential for inhibition of NK cell functions. Different effector mechanisms used by CD4+CD25+ Treg in the same or different situations and in the presence of different types or activation states of cells to be suppressed are easily conceivable, and at least four have already been described to date: contact-mediated, cytokine-independent inhibition in vitro on conventional T cells; cytokine (TGF-ß and/or IL-10)-mediated inhibition in several in vivo models; inhibition of NK cells by membrane-bound TGF-ß; and direct cytotoxicity. In addition, inherent characteristics of the target cells (T vs. NK; resting vs. activated) might determine susceptibility or resistance to one or another mechanism of Treg-mediated suppression.
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TREG-MEDIATED NK CELL INHIBITION ESSENTIALLY TARGETS NKG2D |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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The three tumor cell lines, 3LL, B16-Rae1, and RMA-S-Rae1ß, used by Smyth et al. [38
] for in vivo tumor rejection experiments, express a ligand for NKG2D, and thus, it is again this receptor that might be responsible for the increased tumor growth control observed after Treg depletion. Parental B16 cells gave elevated numbers of lung metastases, and parental RMA-S cells grew s.c. at a similar rate, irrespective of whether Treg had been depleted. As already discussed above, similar observations were made in vitro [38
], as parental cell lines were less sensitive to NK cell-mediated killing, and their lysis levels were not influenced by the presence or absence of Treg.
Altogether, these results suggest that the suppressive effect of CD4+CD25+ Treg on the cytotoxicity of NK cells is for a major part a result of down-regulation of NKG2D mediated by TGF-ß [37 ] and that Treg seem to rather selectively inhibit NKG2D-mediated NK cell cytotoxicity [37 , 38 ]. However, available data in this regard are, to some extent, contradictory: Whereas Ghiringhelli et al. [37 ] did observe Treg- and by extension, TGF-ß-mediated down-regulation of NKG2D but not of the NCR NKp30, Castriconi et al. [57 ] previously reported that NKG2D and NKp30, but not NKp46, are down-modulated by TGF-ß and that this leads to reduced NK cell cyotoxicity toward appropriate targets. Similarly, Lee et al. [58 ] have shown that in cancer patients, impairment of NK cell cytotoxic activity might be explained by elevated TGF-ß production, inducing down-regulation of NKG2D. In contrast, although the results of Smyth et al. [38 ] about the selective inhibition of NKG2D-dependent NK cell functions by Treg are pretty clear, these authors did not observe a reduction of NKG2D expression levels on NK cells exposed to activated Treg. Therefore, it is unlikely that proliferation of NK cells in vivo is inhibited through an action of Treg on NKG2D, as NKG2D engagement alone also does not stimulate NK cell proliferation [59 ]. An additional Treg effector mechanism on NK cells might therefore be postulated.
However, it is known that NKG2D is also down-regulated by prolonged exposure to its ligands, with the consequence of reduced cytotoxicity but increased IFN- production [60
], and thus, the presence of ligands might also be able to influence the functions of NKG2D. As Smyth et al. [38
] pointed out, an effect of TGF-ß on NKG2D signaling (rather than surface-expression levels) and/or on ligand expression on surrounding cells might likewise play a role, and indirect effects of Treg on NK cells should not yet be excluded from consideration. Indeed, in the papers of Romagnani et al. [41
] and Shimizu et al. [43
], Treg-mediated inhibition of NK cells seemed to be a consequence of the suppression of IL-2 (acting as a growth and activation factor for NK cells) production by conventional CD4+CD25– T lymphocytes, rather than a direct action on NK cells.
It has been described [59 ] that NKG2D alone is insufficient to trigger cytotoxicity of resting human NK cells (whereas it can activate resting mouse NK cells on its own) but can costimulate NK cells when other AR are engaged in parallel. This might be the reason why, in the complimentary data shown by Ghiringhelli et al. [37 ], lysis of K562 and even more significantly, that of GIST882 by resting human NK cells is not abolished completely but only diminished in the presence of an anti-NKG2D antibody. Other AR must play a role, as also illustrated by the in vitro findings of Smyth et al. [38 ]. However, it seems as if these other AR would not be affected by the suppressive actions of Treg, and therefore, it could be interesting to investigate this point for defined AR/ligand pairs (other than NKG2D and its counterstructures) under appropriate conditions. It could, in particular, be quite easily checked if Treg influence NK cell-mediated ADCC based on the AR CD16. Such a study would focus on a receptor-ligand system and a NK cell function completely independent of NKG2D.
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ACTIVATED NK CELLS ARE OFTEN RESISTANT TO TREG-MEDIATED INHIBITION |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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production by NK cells induced by IL-2 and IL-15 was not inhibited in the presence of Treg compared with that of conventional T cells, whereas IL-12-dependent IFN- release was suppressed strongly by Treg. The robust IFN- production in response to IL-2 [37
] might be surprising, as IL-2 alone is usually shown not to elicit IFN- release by NK cells [61
, 62
]. Barao et al. [39
], who show that injection of the NK cell-stimulating compound polyinosinic:polycytidylic acid into recipient mice enhanced BMG rejection, even when Treg had not been removed from the recipients, also suggested escape of activated NK cells to Treg-mediated suppression. The molecular mechanism of the "activated NK cell resistance" to Treg has not yet been investigated. NKG2D is of course also expressed on activated NK cells, but maybe other AR, whose expression levels increase or as in the case of NKp44 in human [30 , 31 ], are induced solely upon activation and/or the signaling pathways triggered by the stimulating cytokines, make the difference. In the mouse, the appearance on activated NK cells of an additional isoform of NKG2D paired with the adaptor molecule DAP12 [59 ] might increase the activation to a level such that Treg cannot suppress it efficiently.
In any case, these observations suggest that in the context of an acute immune response, when different types of immune cells respond to "danger" signals, high amounts of NK cell-activating cytokines are released and might be able to over-ride Treg-mediated inhibition, rendering NK cells efficient in first-line defense and in subsequent coordination of the adaptive immune response. However, the finding that cytokine-induced IFN- production by NK cells was inhibited by Treg, only when stimulated by IL-12 but not by other cytokines, is somehow reminiscent of the selective Treg action on NKG2D but not other AR and could reflect that Treg-mediated control of NK cells is far from being a simple process but that the outcome of the encounter of both cell types closely depends on the type of NK cell stimuli and more generally, the cells, cytokines, effector molecules, and danger signals present in the environment. There is presumably a complex network of interactions among Treg, NK cells, and "third parties."
Ghiringhelli et al. [37 ] used resting human Treg in the experiments, showing resistance of activated NK cells to inhibition. It could therefore be interesting to study the outcome of an encounter between human Treg and NK cells when both populations are activated. Would Treg then be more efficient or not, considering that they presumably would express lower amounts of membrane-bound TGF-ß [48 ]?
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CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONCD4+CD25+ TREG INHIBIT NK...CD4+CD25+ TREG INHIBIT NK...THE INHIBITION OF NK...TREG-MEDIATED NK CELL INHIBITION...ACTIVATED NK CELLS ARE...CONCLUDING REMARKSREFERENCES |
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Instead of them, more "sophisticated" in vitro cytotoxicity assays and in vivo tumor or BMG rejection experiments were performed. This might also be related to the fact that NK cells are most often studied in terms of cytotoxicity and cytokine production. Nevertheless, NK cell proliferation was not left behind completely, and besides the experiments of Romagnani et al. [41 ], in the particular context of CD56bright NK cell proliferation in response to plasmacytoid DC and IL-2, the results of Ghiringhelli et al. [37 ] suggest that Treg might well control NK cell numbers under physiological conditions. Although not shown, Smyth et al. [38 ], in contrast, report the absence in Treg-depleted mice of any changes in NK cell numbers and homeostatic, proliferative capacity.
From a finalistic point of view, one might ask why Treg inhibit NK cells. It could be conceivable that permanently "uncontrolled" and "uninhibited" NK cells might be dangerous to the host. This might also be relevant relative to the fact that several types of normal body cells can express NKG2D ligands [59 ] and that Treg-mediated inhibition of NK cells might thus contribute to the regulation of tissue homeostasis. Furthermore, a disease-promoting effect of NK cells in several autoimmune disease models has been described, which may be based on direct cytotoxicity against cells in target tissues, production of cytokines that activate CD4+ T helper cells and macrophages, and induction of maturation of DC [63 ]. Conversely, protective effects of NK cells in autoimmune diseases have also been observed [63 , 64 ] so that the complete picture might be complicated and depend on multiple NK cell-intrinsic and environmental factors. Furthermore, Treg are deficient numerically and functionally in autoimmune diseases [65 ]. They could, in this case, no longer be able to control NK cells efficiently, which might worsen the pathologic processes.
During infectious diseases, Treg are involved in limiting exaggerated responses with the potential for autologous damage but sometimes also inhibit an efficient response [1 , 5 ]. It would be important to study the interactions between NK cells and Treg during different types of viral, bacterial, and parasitic infections, first of all, to better understand their interplay but also, to discover potential, new, therapeutic options improving immune defense mechanisms against infectious agents without shifting to deleterious autoimmunity and this by acting on NK cells, Treg, or both. For example, investigation of the potential effect of Treg on the NCR NKp46 and NKp30 in certain viral infections such as influenza could be promising.
Another field of major interest in this context is cancer. NK cells are frequently, functionally impaired in cancer patients and comparable animal models, whereas Treg rather seem to accumulate in blood and tumors and more generally, to preclude an efficient antitumor response of the host’s immune system [1 , 3 , 66 ]. This deleterious effect might at least in part be based on Treg-mediated inhibition of NK cells [37 , 38 ]. In theory, more efficient anticancer responses might therefore be obtained by removing or inhibiting Treg on the one hand and by stimulating NK cells on the other. Such a double strategy would, however, obviously increase the risk of severe autoimmune phenomena and would therefore need to be carefully explored in animal models. Furthermore, not all potentially interesting molecules seem to be useful. For instance, IL-2 efficiently activates NK cells, but it is also a crucial survival and activating factor for Treg, which would probably compete efficiently for IL-2 as a result of their constitutive expression of CD25. Moreover, increased numbers of highly suppressive CD4+CD25+ Treg develop in IL-2-treated cancer patients [67 ]. A molecule deserving detailed investigation could be GITR, expressed by Treg and activated NK cells and whose triggering suppresses Treg-mediated inhibition [1 , 3 ] but stimulates NK cell cytotoxicity and cytokine production [68 ].
With BMG, the picture is different if one considers an autologous or an allogeneic setting. The results from Barao et al. [39 ] suggest that Treg depletion before autologous BMG could favor hematopoietic reconstitution, whereas the same procedure before an allogeneic graft is likely to be followed by an increased rejection of donor cells (mediated by recipient NK cells).
Finally, besides naturally occurring CD4+CD25+ Treg, there are several other types of Treg whose potential effects on NK cells remain to be studied. It is likely that research will go forward rapidly in this field and that there is more to come about Treg-mediated inhibition of NK cell functions and the precise circumstances under which this regulation takes place or not.
Received June 22, 2006; revised August 2, 2006; accepted August 3, 2006.
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for TH1 priming Nat. Immunol. 5,1260-1265[CrossRef][Medline]
