A role for IFN-gamma in primary and secondary immunity generated by NK cell-sensitive tumor-expressing CD80 in vivo

We have investigated the primary and secondary immunity generated in vivo by a MHC class I-deficient tumor cell line that expressed CD80 (B7-1). CD80 expression enhanced primary NK cell-mediated tumor rejection in vivo and T cell immunity against secondary tumor challenge. CD80 expression enhanced primary NK cell-mediated tumor rejection, and both NK cell perforin and IFN-gamma activity were critical for the rejection of MHC class I-deficient RMA-S-CD80 tumor cells. This primary rejection process stimulated the subsequent development of specific CTL and Th1 responses against Ags expressed by the MHC class I-deficient RMA-S tumor cells. The development of effective secondary T cell immunity could be elicited by irradiated RMA-S-CD80 tumor cells and was dependent upon NK cells and IFN-gamma in the priming response. Our findings demonstrate a key role for IFN-gamma in innate and adaptive immunity triggered by CD80 expression on tumor cells.     

Cutting edge: novel priming of tumor-specific immunity by NKG2D-triggered NK cell-mediated tumor rejection and Th1-independent CD4+ T cell pathway

NKG2D is an activation receptor on NK cells and has been demonstrated as a primary cytotoxicity receptor for mouse NK cells. Primary rejection of class I-deficient RMA-S lymphoma cells expressing the NKG2D ligand, retinoic acid early inducible-1beta, was critically dependent upon NK cell perforin and occurred independently of T cells. NKG2D-triggered NK cell rejection of RMA-S-retinoic acid early inducible-1beta tumor primed a secondary tumor-specific T cell response mediated by both CD4+ and CD8+ T cells in the effector phase. Surprisingly, during the priming phase, CD4+ T cells, but not CD8+ T cells, were also required to generate this secondary T cell immunity; however, T cell priming was independent of Th1 cytokines, such as IFN-gamma and IL-12. These data imply a novel pathway for priming T cell immunity, that is, stimulated upon NK cell-mediated cytotoxicity of NKG2D ligand-expressing tumor cells, dependent upon CD4+ T cells in the primary phase, and independent of conventional Th1-type immunity.     

T cells gene-engineered with DAP12 mediate effector function in an NKG2D-dependent and major histocompatibility complex-independent manner

NKG2D is an important activating/co-stimulatory receptor harnessed by NK and T cells in immune surveillance. In contrast to NK cells, T cells fail to express the activation-signaling molecule DAP12 even when activated and, therefore, ligation of NKG2D alone is insufficient to induce T cell cytolytic function. To test whether we could endow T cells with NK cell-like effector function, we have engineered DAP12 into T cells by retroviral transduction (T-DAP12). T-DAP12 cells were demonstrated to specifically secrete interferon-gamma following receptor ligation and to mediate potent and specific lysis of the NKG2D ligand (NKG2D-L) (Rae-1beta) expressing MHC class I-deficient and class I-sufficient tumors. To circumvent the inability of T-DAP12 cells to proliferate following NKG2D ligation by Rae-1beta expressing tumors, DAP12 was engineered into OT-1 cells with an endogenous T cell receptor specific for chicken ovalbumin peptide (amino acids 257-264). Importantly, following a period of proliferation through endogenous T cell receptor ligation, OT-1-DAP12 cells retained specificity against NKG2D-L expressing major histocompatibility complex class I-deficient tumor. In adoptive transfer experiments, T-DAP12 cells enhanced the survival of NK cell-depleted RAG-1-deficient mice inoculated with RMA-S-Rae-1beta but not parental RMA-S tumors. Overall, this study demonstrated the significant potential of suppressing tumors and other cellular targets expressing NKG2D-L by endowing T cells with innate NK cell-like function.     

A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells

NK cells provide a line of defense against tumors and virus-infected cells that have lost the expression of one or more MHC class I isoforms. Here, we investigate whether inhibitors of apoptosis can block the rejection of tumors mediated by NK cells, by introducing the long form of Fas-associated death domain-like IL-1beta-converting enzyme-associated inhibitory protein (FLIP(L)) and poxvirus cytokine response modifier A (CrmA) into the MHC class I-deficient T lymphoma cell line RMA-S. RMA-S cells do not normally express Fas in vitro, and it was previously postulated that the rejection of these tumors by NK cells is strictly perforin dependent. We show that perforin-deficient NK cells directly mediate Fas up-regulation on RMA-S cells and thereafter kill the cells in a Fas-dependent manner, and that RMA-S FLIP(L) and RMA-S CrmA are protected from such killing. When injected in immunocompetent recipients, RMA-S cells up-regulate Fas, rendering in vivo-passed mock-transduced cells sensitive to Fas-mediated apoptosis. Moreover, RMA-S FLIP(L) and RMA-S CrmA cells establish aggressive tumors, in contrast to RMA-S mock cells that are rejected. These results demonstrate that FLIP(L) and CrmA function as tumor progression factors by protecting MHC class I-deficient tumors from rejection mediated by NK cells. Moreover, our data indicate that death receptor-mediated apoptosis has a more prominent role in the clearance of NK-sensitive tumors than previously suggested.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

IFN-gamma production and cytotoxicity of IL-2-activated murine NK cells are differentially regulated by MHC class I molecules

Activation of NK cells by target cells leads to cytotoxicity as well as production of various cytokines including IFN-gamma. MHC class I molecules on target cells regulate NK cytotoxicity. However, little is known about the regulation of IFN-gamma production by NK cells. We examined the production of IFN-gamma in individual murine NK cells stimulated with tumor cell lines by flow cytometric analysis of intracellular IFN-gamma. Among several tumor lines tested, the rat basophilic leukemia line RBL-1 induced particularly high level of IFN-gamma production in IL-2-activated NK cells, whereas other lines, including the prototypic NK target YAC-1, induced very low or no IFN-gamma production. Transfection of murine classical MHC class I molecules into RBL-1 cells substantially inhibited IFN-gamma production. This inhibition of IFN-gamma production by MHC class I was independent of Ly-49 or CD94/NKG2A expression on NK cells. These results indicate that some target cells directly stimulate IL-2-activated NK cells and induce IFN-gamma production, but the requirements for the induction of IFN-gamma production seem different from those for NK cytotoxicity. Furthermore, similar to NK cytotoxicity, induction of IFN-gamma production is inhibited by MHC class I on stimulating cells. However, the MHC class I-specific receptors inhibiting IFN-gamma production are different from those for NK cytotoxicity.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.     

Ex vivo expansion of non-MHC-restricted cytotoxic effector cells as adoptive immunotherapy for myeloma

BACKGROUND: PBMC can be expanded ex vivo into aggressive cytotoxic effector cells (CEC) comprising T, NK and NKT cells. We identified the phenotype, cytotoxicity and mechanisms of killing of these CEC. METHODS: CY- and G-CSF-mobilized PBMC from myeloma patients were placed in Aim-V serum-free medium, IL-2 (50 IU/mL) and OKT-3 (50 ng/mL). Cytotoxicity was evaluated by selectively blocking the TCR, MHC class I or NKG2D receptor. RESULTS: The CEC expanded three-fold by day 7 and aggressively lysed myeloma cells (41.9%) compared with day 0 (4%; P=0.012). CD8+ CD56+ NKT cells performed the majority of lysis. The CD8+ cells greatly increased NKG2D expression during culture (P=0.005). Cytotoxicity correlated with target NKG2D ligand expression (P=0.0002). Blocking the TCR or MHC class I did not affect cytotoxicity (P>0.22). CD8+ cell-mediated lysis dropped 48% when the NKG2D receptor was blocked. Day 7 CEC aggressively lysed myeloma cells in an MHC- and non-MHC-restricted fashion, through the NKG2D receptor. DISCUSSION: Because MHC expression is often down-regulated on tumor cells and the NKG2D ligands are generally specific to malignant cells, the adoptive transfer of CEC that kill through different pathways may circumvent tumor-resistant mechanisms and improve outcomes.     

Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.     

Oral administration of high molecular mass poly-gamma-glutamate induces NK cell-mediated antitumor immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-gamma-glutamate (gamma-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa gamma-PGA or beta-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-gamma secretion in mice treated with higher molecular mass gamma-PGA (2000 kDa) vs those treated with lower molecular mass gamma-PGA (10 or 100 kDa) or beta-glucan. We then examined the effect of oral administration of 10- or 2000-kDa gamma-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass gamma-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that gamma-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-gamma knockout mice. Taken together, we demonstrated that oral administration of high molecular mass gamma-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12

NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.     

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.     

Macrophages help NK cells to attack tumor cells by stimulatory NKG2D ligand but protect themselves from NK killing by inhibitory ligand Qa-1

Natural killer (NK) cells and their crosstalk with other immune cells are important for innate immunity against tumor. To explore the role of the interaction between NK cells and macrophages in the regulation of anti-tumor activities of NK cells, we here demonstrate that poly I:C-treated macrophages increased NK cell-mediated cytotoxicity against target tumor cells in NKG2D-dependent manner. In addition, IL-15, IL-18, and IFN-β secreted by poly I:C-treated macrophages are also involved in NKG2D expression and NK cell activation. Interestingly, the increase in expression of NKG2D ligands on macrophages induced a highly NK cell-mediated cytotoxicity against tumor cells, but not against macrophages themselves. Notably, a high expression level of Qa-1, a NKG2A ligand, on macrophages may contribute to such protection of macrophages from NK cell-mediated killing. Furthermore, Qa-1 or NKG2A knockdown and Qa-1 antibody blockade caused the macrophages to be sensitive to NK cytolysis. These results suggested that macrophages may activate NK cells to attack tumor by NKG2D recognition whereas macrophages protect themselves from NK lysis via preferential expression of Qa-1.     

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.     

双歧杆菌脂磷壁酸对B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响

目的探讨双歧杆菌脂磷壁酸（LTA）对黑色素瘤B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响。方法将黑色素瘤B16细胞接种于C57BL／6小鼠皮下,待触及肿块后于荷瘤小鼠皮下注射双歧杆菌LTA。采用MTT、流式细胞术（FCM）、RT-PCR方法分别检测经双歧杆菌LTA处理后B16荷瘤小鼠NK细胞杀伤活性、NK细胞NKG2D受体蛋白表达以及肿瘤组织内Rae-1、H60 mRNA表达的变化。结果与对照组相比,经双歧杆菌LTA处理后,B16荷瘤小鼠的NK细胞杀伤活性增强（P〈0．05）,NK细胞受体NKG2D表达明显增加（P〈0．05）,肿瘤组织Rae-1、H60 mRNA表达上升（P〈0．05）,并具有浓度依赖性。结论双歧杆菌LTA能够增强B16荷瘤小鼠NK细胞的杀伤活性,其机制可能与上调NK细胞受体NKG2D的蛋白表达和肿瘤组织Rae-1、H60 mRNA的表达有关。     

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.