Production of a novel bispecific protein ULBP1×CD19-scFv targeting the NKG2D receptor and CD19 to promote the activation of NK cells.

Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production.

Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rß and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.

T Cells Infiltrating Diseased Liver Express Ligands for the NKG2D Stress Surveillance System.

NK cells, which are highly enriched in the liver, are potent regulators of antiviral T cells and immunopathology in persistent viral infection. We investigated the role of the NKG2D axis in T cell/NK cell interactions in hepatitis B. Activated and hepatitis B virus (HBV)-specific T cells, particularly the CD4 fraction, expressed NKG2D ligands (NKG2DL), which were not found on T cells from healthy controls (p < 0.001). NKG2DL-expressing T cells were strikingly enriched within HBV-infected livers compared with the periphery or to healthy livers (p < 0.001). NKG2D+NK cells were also increased and preferentially activated in the HBV-infected liver (p < 0.001), in direct proportion to the percentage of MICA/B-expressing CD4 T cells colocated within freshly isolated liver tissue (p < 0.001). This suggests that NKG2DL induced on T cells within a diseased organ can calibrate NKG2D-dependent activation of local NK cells; furthermore, NKG2D blockade could rescue HBV-specific and MICA/B-expressing T cells from HBV-infected livers. To our knowledge, this is the first ex vivo demonstration that non-virally infected human T cells can express NKG2DL, with implications for stress surveillance by the large number of NKG2D-expressing NK cells sequestered in the liver.

NKG2D-NKG2D Ligand Interaction Inhibits the Outgrowth of Naturally Arising Low-Grade B Cell Lymphoma In Vivo.

It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms. One of these mechanisms is the NKG2D receptor that recognizes ligands whose expression is induced by cell transformation. In this study, we show that in NKG2D receptor-deficient mice, increasing numbers of B cells begin to express NKG2D ligands as they age. Their absence in wild-type mice suggests that these cells are normally cleared by NKG2D-expressing cells. NKG2D-deficient mice and mice constitutively expressing NKG2D ligands had increased incidence of B cell tumors, confirming that the inability to clear NKG2D ligand-expressing cells was important in tumor suppression and that NKG2D ligand expression is a marker of nascent tumors. Supporting a role for NKG2D ligand expression in controlling the progression of early-stage B cell lymphomas in humans, we found higher expression of a microRNA that inhibits human NKG2D ligand expression in tumor cells from high-grade compared with low-grade follicular lymphoma patients.

NKG2D expression by CD8+ T cells contributes to GVHD and GVT effects in a murine model of allogeneic HSCT.

In allogeneic hematopoietic stem cell transplantation (HSCT), controlling graft-versus-host disease (GVHD) while maintaining graft-versus-tumor (GVT) responses is of critical importance. Using a mouse model of allogeneic HSCT, we hereby demonstrate that NKG2D expression by CD8(+) T cells plays a major role in mediating GVHD and GVT effects by promoting the survival and cytotoxic function of CD8(+) T cells. The expression of NKG2D ligands was not induced persistently on normal tissues of allogeneic HSCT-recipient mice treated with anti-NKG2D antibody, suggesting that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8(+) T cells to regain their GVT function. Indeed, short-term treatment with anti-NKG2D antibody restored GVT effects while maintaining an attenuated GVHD state. NKG2D expression was also detected on CD8(+) T cells from allogeneic HSCT patients and trended to be higher in those with active GVHD. Together, these data support a novel role for NKG2D expression by CD8(+) T cells during allogeneic HSCT, which could be potentially therapeutically exploited to separate GVHD from GVT effects.

Role of NKG2D, DNAM-1 and natural cytotoxicity receptors in cytotoxicity toward rhabdomyosarcoma cell lines mediated by resting and IL-15-activated human natural killer cells.

Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.

NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus).

The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

NKG2D blockade inhibits poly(I:C)-triggered fetal loss in wild type but not in IL-10-/- mice.

Infection and inflammation can disturb immune tolerance at the maternal-fetal interface, resulting in adverse pregnancy outcomes. However, the underlying mechanisms for detrimental immune responses remain ill defined. In this study, we provide evidence for immune programming of fetal loss in response to polyinosinic:polycytidylic acid (polyI:C), a viral mimic and an inducer of inflammatory milieu. IL-10 and uterine NK (uNK) cells expressing the activating receptor NKG2D play a critical role in poly(I:C)-induced fetal demise. In wild type (WT) mice, poly(I:C) treatment induced expansion of NKG2D(+) uNK cells and expression of Rae-1 (an NKG2D ligand) on uterine macrophages and led to fetal resorption. In IL-10(-/-) mice, NKG2D(-) T cells instead became the source of fetal resorption during the same gestation period. Interestingly, both uterine NK and T cells produced TNF-α as the key cytotoxic factor contributing to fetal loss. Treatment of WT mice with poly(I:C) resulted in excessive trophoblast migration into the decidua and increased TUNEL-positive signal. IL-10(-/-) mice supplemented with recombinant IL-10 induced fetal loss through NKG2D(+) uNK cells, similar to the response in WT mice. Blockade of NKG2D in poly(I:C)-treated WT mice led to normal pregnancy outcome. Thus, we demonstrate that pregnancy-disrupting inflammatory events mimicked by poly(I:C) are regulated by IL-10 and depend on the effector function of uterine NKG2D(+) NK cells in WT mice and NKG2D(-) T cells in IL-10 null mice.

Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells.

In humans, the interaction of the natural killer group 2 member D (NKG2D)-activating receptor on natural killer (NK) and CD8(+) T cells with its major histocompatibility complex class I-related chain (MIC) and UL16 binding protein (ULBP) ligands (NKG2DLs) promotes recognition and elimination of stressed cells, such as tumor or infected cells. Here, we investigated the capacity of HIV-1 to modulate NKG2DL expression and escape NGK2D-mediated immunosurveillance. In CD4(+) T lymphocytes, both cell surface expression and release of MICA, MICB, and ULBP2 were up-regulated >2-fold by HIV-1 infection. In HIV-infected CD4(+) T lymphocytes or Jurkat T-cell lines, increased shedding of soluble NKG2DLs (sNKG2DLs) was impaired by a matrix metalloproteinase inhibitor (MMPI). Moreover, naive HIV(+) patients displayed increased plasma sMICA and sULBP2 levels and reduced NKG2D expression on NK and CD8(+) T cells compared to patients receiving highly active antiretroviral therapy (HAART) or healthy donors. In individual patients, HAART uptake resulted in the drop of sNKG2DL and recovery of NKG2D expression. Finally, sNKG2DLs in patients' plasma down-regulated NKG2D on NK and CD8(+) T cells and impaired NKG2D-mediated cytotoxicity of NK cells. Thus, NKG2D detuning by sNKG2DLs may promote HIV-1 immune evasion and compromise host resistance to opportunistic infections, but HAART and MMPI have the potential to avoid such immune dysfunction.

Involvement of an NKG2D ligand H60c in epidermal dendritic T cell-mediated wound repair.

Dendritic epidermal T cells (DETCs) found in mouse skin are NKG2D-positive γδ T cells involved in immune surveillance and wound repair. It is assumed that the interaction of an NKG2D receptor on DETCs and an MHC class I-like NKG2D ligand on keratinocytes activates DETCs, which then secrete cytokines promoting wound repair. However, direct evidence that DETC activation through NKG2D signaling promotes wound repair is not available. In the present study, we generated mAbs for an NKG2D ligand H60c previously suggested to be expressed specifically on skin keratinocytes. Local administration of H60c-specific mAb inhibited activation of DETCs and significantly delayed wound repair. Likewise, administration of NKG2D-specific mAb impaired wound repair to a similar extent. The delay in wound closure resulting from the blockade of the NKG2D pathway was comparable to that observed in γδ T cell-deficient mice. These results indicate that H60c/NKG2D interactions play a critical role in wound repair. Reassessment of binding affinities showed that H60c monomers bind to NKG2D with affinity (K(d) = 26 ± 3.2 nM) comparable to those of other high-affinity NKG2D ligands. H60c is transcribed not only in skin but also in tissues such as tongue and female reproductive tract known to contain epithelium-resident γδ T cells expressing invariant TCRs, suggesting a more general role for H60c in the maintenance of epithelial integrity.

Negative regulation of NKG2D expression by IL-4 in memory CD8 T cells.

IL-4 is one of the main cytokines produced during Th2-inducing pathologies. This cytokine has been shown to affect a number of immune processes such as Th differentiation and innate immune responses. However, the impact of IL-4 on CD8 T cell responses remains unclear. In this study, we analyzed the effects of IL-4 on global gene expression profiles of Ag-induced memory CD8 T cells in the mouse. Gene ontology analysis of this signature revealed that IL-4 regulated most importantly genes associated with immune responses. Moreover, this IL-4 signature overlapped with the set of genes preferentially expressed by memory CD8 T cells over naive CD8 T cells. In particular, IL-4 downregulated in vitro and in vivo in a STAT6-dependent manner the memory-specific expression of NKG2D, thereby increasing the activation threshold of memory CD8 T cells. Furthermore, IL-4 impaired activation of memory cells as well as their differentiation into effector cells. This phenomenon could have an important clinical relevance as patients affected by Th2 pathologies such as parasitic infections or atopic dermatitis often suffer from viral-induced complications possibly linked to inefficient CD8 T cell responses.

NKG2D blockade attenuated cardiac allograft vasculopathy in a mouse model of cardiac transplantation.

A previous paper has reported that blockade of NKG2D was effective in protecting allograft in murine models of cardiac transplantation, but the mechanism of NKG2D blockade on attenuated cardiac allograft vasculopathy (CAV) was still unknown. In our current study, we found that wild-type recipients treated with anti-NKG2D monoclonal antibody (mAb) plus cytotoxic T lymphocyte antigen (CTLA)-4-immunoglobulin (I)g showed prolonged allograft survivals (>90 days, P < 0·001) significantly and attenuated CAV. These in-vivo results correlated with reduced alloantibody production, low expression of interleukin (IL)-17 and IL-6, while infiltration of regulatory T cells increased. IL-6 administration induced shorter allograft survival and higher CAV grade in CTLA-4-Ig plus anti-NKG2D mAb-treated recipients, whereas IL-17 had no significant effect on allograft survival and CAV grade in CTLA-4-Ig plus anti-NKG2D mAb-treated recipients. Furthermore, the prolonged allograft survival induced by NKG2D blockade was abrogated partially with depletion of regulatory T cells. In conclusion, blockade of NKG2D combined with CTLA-4-Ig attenuated CAV and this effect was associated with lower alloantibody production, inhibited IL-6 expression and enhanced expansion of regulatory T cells.

NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing.

Natural killer (NK) cells are lymphocytes of the innate immune system that have crucial cytotoxic and regulatory roles in adaptive immunity and inflammation. Herein, we consider a role for these cells in corneal wound healing. After a 2-mm central epithelial abrasion of the mouse cornea, a subset of classic NK cells migrated into the limbus and corneal stroma, peaking at 24 hours with an eightfold increase over baseline. Depletion of γδ T cells significantly reduced NK cell accumulation (>70%; P < 0.01); however, in neutrophil-depleted animals, NK cell influx was normal. Isolated spleen NK cells migrated to the wounded cornea, and this migration was reduced by greater than 60% (P < 0.01) by ex vivo antibody blocking of NK cell CXCR3 or CCR2. Antibody-induced depletion of NK cells significantly altered the inflammatory reaction to corneal wounding, as evidenced by a 114% increase (P < 0.01) in neutrophil influx at a time when acute inflammation is normally waning. Functional blocking of NKG2D, an activating receptor for NK cell cytotoxicity and cytokine secretion, did not inhibit NK cell immigration, but significantly increased neutrophil influx. Consistent with excessive neutrophil accumulation, NK depletion and blocking of NKG2D also inhibited corneal nerve regeneration and epithelial healing (P < 0.01). Findings of this study suggest that NK cells are actively involved in corneal healing by limiting the innate acute inflammatory reaction to corneal wounding.

NK cells promote type 1 T cell immunity through modulating the function of dendritic cells during intracellular bacterial infection.

Dendritic cells (DC) play a key role in establishing protective adaptive immunity in intracellular bacterial infections, but the cells influencing DC function in vivo remain unclear. In this study, we investigated the role of NK cells in modulating the function of DC using a murine Chlamydia infection model. We found that the NK cell-depleted mice showed exacerbated disease after respiratory tract Chlamydia muridarum infection, which was correlated with altered T cell cytokine profile. Furthermore, DC from C. muridarum-infected NK-depleted mice (NK(-)DC) exhibited a less mature phenotype compared with that of DC from the infected mice without NK depletion (NK(+)DC). NK(-)DC produced significantly lower levels of both IL-12 and IL-10 than those of NK(+)DC. Moreover, NK(-)DC showed reduced ability to direct primary and established Ag-specific Th1 CD4(+) T cell responses in DC-T coculture systems. More importantly, adoptive transfer of NK(-)DC, in contrast to NK(+)DC, failed to induce type 1 protective immunity in recipients after challenge infection. Finally, NK cells showed strong direct enhancing effect on IL-12 production by DC in an NK-DC coculture system, which was partially reduced by blocking NKG2D receptors signaling and virtually abolished by neutralizing IFN-γ activity. The data demonstrate a critical role of NK cells in modulating DC function in an intracellular bacterial infection.

Human microRNA-1245 down-regulates the NKG2D receptor in natural killer cells and impairs NKG2D-mediated functions.

BACKGROUND: NKG2D is an activating receptor expressed by natural killer and T cells, which have crucial functions in tumor and microbial immunosurveillance. Several cytokines have been identified as modulators of NKG2D receptor expression. However, little is known about NKG2D gene regulation. In this study, we found that microRNA 1245 attenuated the expression of NKG2D in natural killer cells. DESIGN AND METHODS: We investigated the potential interactions between the 3'-untranslated region of the NKG2D gene and microRNA as well as their functional roles in the regulation of NKG2D expression and cytotoxicity in natural killer cells. RESULTS: Transforming growth factor-ß1, a major negative regulator of NKG2D expression, post-transcriptionally up-regulated mature microRNA-1245 expression, thus down-regulating NKG2D expression and impairing NKG2D-mediated immune responses in natural killer cells. Conversely, microRNA-1245 down-regulation significantly increased the expression of NKG2D expression in natural killer cells, resulting in more efficient NKG2D-mediated cytotoxicity. CONCLUSIONS These results reveal a novel NKG2D regulatory pathway mediated by microRNA-1245, which may represent one of the mechanisms used by transforming growth factor-ß1 to attenuate NKG2D expression in natural killer cells.

Blockade of NKG2D ameliorates disease in mice with collagen-induced arthritis: a potential pathogenic role in chronic inflammatory arthritis.

OBJECTIVE: To assess the role of the activating receptor NKG2D in arthritis. METHODS: Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS: We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION: Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Intact NKG2D-independent function of NK cells chronically stimulated with the NKG2D ligand Rae-1.

Human tumors frequently express membrane-bound or soluble NK group 2, member D (NKG2D) ligands. This results in chronic engagement of NKG2D on the surfaces of NK and CD8(+) T cells and rapid internalization of the receptor. Although it is well appreciated that this phenomenon impairs NKG2D-dependent function, careful analysis of NKG2D-independent functions in cells chronically stimulated through NKG2D is lacking. Using a mouse model of chronic NKG2D ligand expression, we show that constant exposure to NKG2D ligands does not functionally impair NK cells and CD8(+) T cells in the context of viral infection.

Regulation of immune cell function and differentiation by the NKG2D receptor.

NKG2D is one of the most intensively studied immune receptors of the past decade. Its unique binding and signaling properties, expression pattern, and functions have been attracting much interest within the field due to its potent antiviral and anti-tumor properties. As an activating receptor, NKG2D is expressed on cells of the innate and adaptive immune system. It recognizes stress-induced MHC class I-like ligands and acts as a molecular sensor for cells jeopardized by viral infections or DNA damage. Although the activating functions of NKG2D have been well documented, recent analysis of NKG2D-deficient mice suggests that this receptor may have a regulatory role during NK cell development. In this review, we will revisit known aspects of NKG2D functions and present new insights in the proposed influence of this molecule on hematopoietic differentiation.

Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function.

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1-5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1-5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8(+), and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.