The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells

Previous studies on NK recognition of HLA-G1 employed as targets 721.221 transfectants (.221-G1) that unknowingly co-expressed the HLA-E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re-evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA-G1 by NK clones. We employed as targets .221-G1 cells and a surface HLA-E-negative transfectant, .221-G1(E_neg), generated by site-directed mutagenesis of the HLA-G1 leader sequence. The antagonistic effects of receptor- (i.e. CD94/NKG2A, ILT2) and ligand-specific mAb (i.e. HLA-G, HLA-E) were assessed. In addition, binding of an ILT2-Ig fusion protein to the .221-AEH, expressing only HLA-E, and the .221-G1(E_neg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: the CD94/NKG2 interaction with HLA-E, and the engagement of the ILT2(LIR1) receptor by HLA-G1 molecules.     

The CD94/NKG2 family of receptors

Immune responses must be tightly regulated to avoid hyporesponsiveness on one hand or excessive inflammation and the development of autoimmunity (hyperresponsiveness) on the other hand. This balance is attained through the throttling of activating signals by inhibitory signals that ideally leads to an adequate immune response against an invader without excessive and extended inflammatory signals that promote the development of autoimmunity. The CD94/NKG2 family of receptors is composed of members with activating or inhibitory potential. These receptors are expressed predominantly on NK cells and a subset of CD8+T cells, and they have been shown to play an important role in regulating responses against infected and tumori genic cells. In this review, we discuss the current knowledge about this family of receptors, including ligand and receptor interaction, signaling, membrane dynamics, regulation of gene expression and their roles in disease regulation, infections, and cancer, and bone marrow transplantation.     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR alphabeta lymphocytes

A subset of CD8(+) T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8(+) T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7(+) memory cells and CCR7(-) effector cells. Since NKG2A can only be induced on naive CD8(+) T cells while CD94(-) memory cells are refractory, it is likely that commitment to the CD94:NKG2A(+) subset occurs during the first encounter with antigen. CCR7(+)CD94:NKG2A(+) T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-gamma. These cells occur as a separate CD94:NKG2A(+) T cell lineage with a distinct TCR repertoire that differs from that of the other CD8(+)CD94(-) T cells activated in situ.     

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.     

Association of CD94/NKG2A, CD94/NKG2C, and its ligand HLA-E polymorphisms with Behcet's disease

Inhibitory CD94/NKG2A and activating CD94/NKG2C receptors are expressed on natural killer, CD4, and CD8 T cells and recognize human leukocyte antigen (HLA)-E, resulting in the modulation of cytotoxic activity and cytokine production. An imbalance in cytotoxic activity and cytokine production has been implicated in Behcet's disease (BD). The results of this study showed that the NKG2A c.-4258*C, c.338-90*G, and CD94 c.-134*T alleles (P= 0.015, OR = 0.8; P < 0.0001, OR = 0.5; and P= 0.034, OR = 0.8, respectively) were associated with decreased risk and that NKG2A c.284-67_-62del, c.1077*C, and the activating receptor, NKG2C c.305*T were not associated with 345 patients with BD. But a significant difference in NKG2C c.305*T was detected among BD patients with ocular lesions and arthritis (P < 0.0001, OR = 2.1 and P= 0.0001, OR = 1.8, respectively). We already showed in our previous research that HLA-E*0101 also appears to contribute to a reduction in risk through the inhibitory CD94/NKG2A-mediated immune response. This result led us to the analyses of the combined risk of the HLA-E and the NKG2A for BD. Individuals harboring HLA-E*0101, NKG2A c.-4258*C, and c.338-90*G evidenced a reduced risk of BD compared with healthy controls (21.1% vs 40.1%, P < 0.0001, OR = 0.4). By way of contrast, individuals without the HLA-E*0101, NKG2A c.-4258*C, and c.338-90*G alleles evidenced a twofold increased risk of BD (P= 0.014, OR = 2.0). Individuals without HLA-E*0101, NKG2A c.-4258*G/*G, and c.338-90*G evidenced a 4.8-fold increase in BD risk (P= 0.0002, OR = 4.8). Although the effects of these single nucleotide polymorphisms (SNPs) remain unclear, our results indicate that the SNPs of the inhibitory receptor CD94/NKG2A and its haplotypes, as well as its ligand HLA-E, are associated with BD immune systems.     

HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex

The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell-mediated lysis. The nonclassical HLA class I molecule HLA-G may mediate protection from NK cells. NK cells are known to express a number of HLA class I-specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I-negative 221 cell line either untransfected or transfected with HLA-G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A-negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA-G-specific inhibitory NKR, and that HLA-G-specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94⁺/NKG2A^? phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA-G specificity, thus suggesting the existence of a different, still unknown NKR.     

MS患者CD94/NKG2A-bright和CD94/NKG2A-dim NK细胞亚群对IFN-β的差异性反应

目的： 分析多发性硬化（MS）进展型患者不同表型NK细胞亚群对临床主要治疗方法的反应性差异.方法： 分离患者外周血中的NK细胞, 以流式细胞术根据表面抑制性受体CD94/NKG2A表达情况分为两个亚群CD94/NKG2A-bright和CD94/NKG2A-dim.分别加入IFN-β, 测定两个亚群表面CD94/NKG2A变化及细胞增殖, 同时检测两种亚群分泌IL-10和TGF-β情况.结果： CD94/NKG2A阳性表达的NK细胞占25.5%, 其中CD94/NKG2A-bright和CD94/NKG2A-dim分别占其中的23.6%和76.4%.加入IFN-β, CD94/NKG2A-bright组增殖率明显低于CD94/NKG2A-dim组, CD94/NKG2A表达峰度变化不大.CD94/NKG2A-dim组中CD94/NKG2A表达显著增加.两个亚群分泌的IL-10和TGF-β与未刺激组相比, 均有明显差异.CD94/NKG2A-bright和CD94/NKG2A-dim组间亦有明显差异.结论： IFN-β通过诱导NK细胞CD94/NKG2A表达在非特异免疫系统中抑制NK细胞; 同时刺激IL-10 和TGF-β分泌进一步发挥对免疫系统的抑制.CD94/NKG2A-bright和CD94/NKG2A-dim对IFN-β反应有差异性.     

NKG2C zygosity influences CD94/NKG2C receptor function and the NK‐cell compartment redistribution in response to human cytomegalovirus

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK‐cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV‐seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C⁺ NK‐cell numbers was observed in HCMV⁺ children. We have assessed the influence of NKG2C gene dose on the NK‐cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C⁺ NK‐cell numbers in HCMV⁺ subjects, independently of HLA‐E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa²⁺ influx), degranulation, and IL‐15‐dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C^+/+ and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady‐state NKG2C⁺ NK‐cell numbers, further supporting an active involvement of the receptor in the HCMV‐induced reconfiguration of the NK‐cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention.     

The CD94/NKG2C killer lectin-like receptor constitutes an alternative activation pathway for a subset of CD8+ T cells

The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.     

The role of TCR stimulation and TGF-beta in controlling the expression of CD94/NKG2A receptors on CD8 T cells

Following antigen recognition, murine CD8 T cells express CD94/NKG2A receptors. Our results show that this up-regulation occurs rapidly in vitro and is accompanied by an approximately 8-fold increase in CD94 and approximately 125-fold increase in NKG2A mRNA. In contrast, only a twofold increase in NKG2C mRNA is noted. The addition of TGF-beta, but not IL-10, IL-12 or IL-15, leads to a further increase in cell membrane expression of these receptors, as well as a approximately 6-fold increase in mRNA for both chains. TGF-beta also increases CD94/NKG2A expression on memory CD8 T cells that are re-exposed to antigen. The effect of TGF-beta on increasing CD94/NKG2A expression on both naive and memory CD8 T cells occurs only when there is a concurrent stimulation through the TCR. In contrast, TGF-beta does not increase expression of CD94/NKG2A on resting or activated NK cells. We also show by using purified CD8 T cells, that TGF-beta acts directly on these cells. These results implicate a role for both antigen and TGF-beta in increasing expression of inhibitory CD94/NKG2A receptors on CD8 T cells.     

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.     

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice

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CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A,but lacking inhibitory killer Ig-like receptors

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.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Changes in Endometrial Natural Killer Cell Expression of CD94, CD158a and CD158b are Associated with Infertility

Problem  Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility.
Method of study  NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples.
Results  While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation.
Conclusion  In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.     

Implication for the CD94/NKG2A-Qa-1 system in the generation and function of ocular-induced splenic CD8+ regulatory T cells

The injection of antigen into the anterior chamber (AC) inducesthe production of antigen-specific splenic CD8⁺ regulatory Tcells (Tregs) /suppressor T cells that perform the local suppressionof delayed-type hypersensitivity (DTH) responses. Because CD94/NKG2A-Qa-1-dependentinteractions have been implicated in CD8⁺ Treg-mediated immunesuppression and DBA/2J mice are deficient in CD94/NKG2R, wehave utilized these mice to test the hypothesis that the CD94/NKG2A-Qa-1system is essential to the induction and immunosuppressive functionof CD8⁺ Tregs in anterior chamber-associated immune deviation(ACAID). We show that: (i) neither ACAID-mediated suppressionof DTH to ovalbumin nor splenic Tregs/suppressor T cells wasinduced in DBA/2J mice that received an injection of antigeninto the AC; (ii) splenic CD8⁺ Tregs from ACAID-induced DBA/2NCrmice suppressed the initiation of DTH when transferred to DBA/2Jmice; (iii) following injection of antigen into the AC, intravenousadministration of splenocytes or Peripheral Blood MononuclearCells (PBMC) isolated from DBA/2NCr but not from DBA/2J micetransferred suppression of DTH to DBA/2NCr mice; (iv) antibodiesto CD94/NKG2A reduced the ACAID CD8⁺ T cell-mediated suppressionof DTH and (v) The deficiency of such immune regulation in DBA/2Jmice also correlated with a decreased number of Qa-1^b+ B cells,F4/80⁺ cells, a deficient number of CD94/NKG2AR and Qa-1 tetramerbinding by CD8⁺ T cells. These results demonstrate that defectiveACAID in DBA/2J mice involves multiple regulatory lesions resultingin a lack of induction of a CD8⁺ Treg response and possiblydefective CD94/NKG2A-dependent suppression of peripheral cell-mediatedimmunity.     

Natural killer cell and hepatic cell interaction via NKG2A leads to dendritic cell-mediated induction of CD4 CD25 T cells with PD-1-dependent regulatory activities

Natural killer (NK) cells have the ability to control dendritic cell (DC)-mediated T cell responses. However, the precise mechanisms by which NK receptor-mediated regulation of NK cells determines the magnitude and direction of DC-mediated T cell responses remain unclear. In the present study, we applied an in vitro co-culture system to examine the impact of NK cells cultured with hepatic cells on DC induction of regulatory T cells. We found that interaction of NK cells and non-transformed hepatocytes (which express HLA-E) via the NKG2A inhibitory receptor resulted in priming of DCs to induce CD4(+) CD25(+) T cells with regulatory properties. NKG2A triggering led to characteristic changes of the cytokine milieu of co-cultured cells; an increase in the transforming growth factor (TGF)-beta involved in the generation of this specific type of DC, and a decrease in the tumour necrosis factor-alpha capable of antagonizing the effect of TGF-beta. The regulatory cells induced by NK cell-primed DCs exert their suppressive actions through a negative costimulator programmed death-1 (PD-1) mediated pathway, which differs from freshly isolated CD4(+) CD25(+) T cells. These findings provide new insight into the role of NK receptor signals in the DC-mediated induction of regulatory T cells.