Author response: Endothelial cells express NKG2D ligands and desensitize antitumor NK responses

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo. https://doi.org/10.7554/eLife.30881.001 eLife digest White blood cells called “natural killer cells” are part of the first line of immune defense. Often called NK cells for short, one job of these cells is to help prevent cancer by killing tumor cells. If an NK cell spots a tumor cell, it must become energized so that it can deliver the killing blow, which comes in the form of a packet of cell-killing “cytotoxic” granules. Yet tumor cells look very similar to healthy cells, and NK cells must be able to tell the difference to be effective. Molecules on the outer surface of the NK cell control how the cell recognizes tumors, and deliver the signals the cell needs to become energized. One of these surface molecules is called NKG2D. It interacts with “partner” molecules found on the surface of cancer cells and tells the NK cell to attack. These partner molecules are not usually found on healthy cells, helping the immune system to tell the difference. After NKG2D interacts with its partner molecules, it moves inside the NK cell. This makes the cell less able to become energized. If the NK cells do not encounter any partner molecules in healthy mice, blocking the interactions should have no effect on NKG2D levels. But now, Thompson et al. find that blocking one of these interactions increased the levels of NKG2D on the surface of NK cells in healthy mice. Further experiments revealed that NK cells in mice constantly encounter an NKG2D partner molecule called RAE-1ε. A search for the source of RAE-1ε in healthy mice pointed to blood vessels inside the lymph nodes. NK cells pass through theses organs as part of their normal path around the body. Thompson et al. also saw that NK cells from healthy mice were less responsive than NK cells from mutant mice that lacked RAE-1ε. As a result of their encounters with RAE-1ε in healthy mice, the NK cells were less able to kill tumor cells. Blocking the interaction between NKG2D and RAE-1ε in mice re-energized their NK cells. More cells were able to enter tumors in these mice and the cells became better at killing tumors. Together these findings increase the current understanding of the biological processes that control NK cells. Further research may lead to new treatments for diseases like cancer. But first, scientists need to find out whether NK cells behave in the same way in humans as they do in mice. If so, developing ways to block the interaction could re-energize human NK cells to better kill cancer cells. https://doi.org/10.7554/eLife.30881.002 Introduction Natural Killer (NK) cells are key effectors in the immune response to pathogens and tumors (Vivier et al., 2008). NK cells respond to infected or transformed cells by releasing cytotoxic granules and anti-tumor cytokines such as interferon-γ (IFNγ) (Vivier et al., 2008; Marcus et al., 2014). NK cells recognize unhealthy cells using an array of cell surface receptors (Vivier et al., 2011; Marcus et al., 2014; Moretta et al., 2014; Morvan and Lanier, 2016). These receptors transmit activating or inhibitory signals upon binding cognate ligands on the target cell, and the net balance of these signals dictates whether the NK cell response is triggered. Tumors are often recognized and killed by NK cells in vitro and in vivo because cancer cells tend to upregulate ligands for activating receptors and downregulate ligands for inhibitory receptors (Waldhauer and Steinle, 2008; Marcus et al., 2014). The responsiveness of NK cells to a given stimulus is dynamically tuned by the steady-state receptor-ligand interactions experienced by the NK cells (Joncker and Raulet, 2008; Brodin et al., 2009; Joncker et al., 2009; Joncker et al., 2010; Shifrin et al., 2014). Increases in steady-state stimulation cause NK cells to compensate by adopting a less responsive state (Joncker et al., 2010; Kadri et al., 2016) – a process that will be referred to here as ‘desensitization’ – whereas NK cells receiving lower steady-state levels of stimulation exhibit a state of heightened responsiveness to acute activation. For example, the Ly49 family of inhibitory receptors on NK cells are known to engage host MHC I molecules at steady state, and this interaction is important for regulating NK responsiveness. Mice lacking MHC I molecules or inhibitory Ly49 receptors show dramatically weaker NK responses to a wide variety of acute stimulatory signals in vitro and in vivo (Liao et al., 1991; Fernandez et al., 2005; Kim et al., 2005; Anfossi et al., 2006; Brodin et al., 2009; Joncker et al., 2010). Desensitization may prevent NK cells from effecting autoreactivity and enable them to adjust to different tissue milieus, and mature NK cells can alter their responsiveness upon encountering a new MHC I environment (Joncker and Raulet, 2008; Elliott et al., 2010; Joncker et al., 2010; Narni-Mancinelli et al., 2013). These dynamics are relevant for antitumor responses, as NK cells in WT mice become desensitized when they infiltrate MHC I-deficient tumors but not when they infiltrate matched MHC-I-positive tumors (Ardolino et al., 2014). Similarly, humans receiving HLA-mismatched bone marrow show altered NK responses that match the trends described in mice (Boudreau et al., 2016). It is presumed that steady-state interactions between MHC I and Ly49 receptors prevent NK desensitization by inhibiting steady-state signals from activating receptors. Indeed, transgenic overexpression of NK activating ligands causes NK desensitization (Oppenheim et al., 2005; Wiemann et al., 2005; Sun and Lanier, 2008; Tripathy et al., 2008), but the endogenous receptor-ligand systems that transmit these activating signals to NK cells in healthy WT animals remain incompletely defined. In humans, activating KIR appear to be one such endogenous signal involved in steady-state NK cell tuning (Fauriat et al., 2010). In mice, the activating receptor NKp46 may contribute to NK desensitization because NKp46-KO animals showed heightened NK responses to stimulation in one report (Narni-Mancinelli et al., 2012), although not in another (Sheppard et al., 2013). SLAM receptors are also reported to regulate NK responsiveness in some contexts (Chen et al., 2016) (Veillette, 2010). Very little is understood about which host cell types are responsible for engaging NK cells to regulate responsiveness. A recent study using β2M-KO bone marrow chimeras suggested that MHC-I-deficient nonhematopoietic cells may play a larger role than MHC-I-deficient hematopoietic cells in desensitizing NK cells, although both may participate (Shifrin et al., 2016). In humans, different studies have implicated HLA molecules on hematopoietic cells (Haas et al., 2011) and nonhematopoietic cells (Cooley et al., 2011) as being critical for tuning. Clearly, much remains to be learned about these processes. Elucidating the receptor-ligand and cellular systems that regulate NK responses in homeostasis and cancer may suggest novel therapeutic strategies. NKG2D is a C-type lectin-like activating receptor expressed by all NK cells and subsets of T cells (Raulet, 2003). NKG2D binds a diverse array of MHC-like proteins. In mice, these include the RAE-1 family (with α, β, γ, δ, and ε isoforms), the H60 family (a, b, c), and MULT1. Human NKG2D ligands include the ULBP family (with isoforms 1–6) and the MICA and MICB proteins (Raulet et al., 2013). Acute NKG2D engagement transmits powerful activating signals through the adaptor molecules DAP10 and DAP12 to drive cytotoxicity and cytokine production (Raulet, 2003). NKG2D ligands are thought to be absent from most healthy cells but can be induced consequent to DNA damage, oncogene signaling, and other stresses associated with cancer and infection (Raulet et al., 2013). Many tumor cells express NKG2D ligands. In tumor transplant and spontaneous cancer models, expression of NKG2D ligand(s) on tumor cells triggers NK activation and protects the host from cancer (Diefenbach et al., 2001; Guerra et al., 2008). Interestingly, several recent studies have shown that NK cells in NKG2D-KO mice are hyper-responsive to stimulation when triggered through other activating receptors (Zafirova et al., 2009; Sheppard et al., 2013). Furthermore, tumor cells engineered to secrete soluble monomeric NKG2D ligands – which block but do not activate NKG2D – increase the responsiveness of tumor-infiltrating NK cells and enhance tumor rejection (Deng et al., 2015). These data suggest that NKG2D may contribute to NK desensitization at steady state or in tumors. In this report, we provide important new findings concerning the cells and molecules that engage NK cells and regulate NK responsiveness, and we clarify the pleiotropic effect of NKG2D on NK activity. Unexpectedly, we show a steady-state interaction between NKG2D and one of its ligands, RAE-1ε, in healthy WT mice. Using bone marrow chimera experiments, we show that non-hematopoietic cells are the primary source of endogenous RAE-1ε. Endothelial cells in lymph nodes were found to be constitutively express RAE-1ε, and RAE-1ε was found to be super-induced on tumor-associated vasculature in transplant and autochthonous cancer models. Importantly, we demonstrate that this interaction between NKG2D and endogenous RAE-1ε desensitizes NK cells and impairs antitumor NK responses and tumor rejection. Results NKG2D is constitutively engaged by endogenous RAE-1ε Cell surface NKG2D ligand expression is usually considered a hallmark of unhealthy cells, but expression on the surface of normal cells in healthy animals has not been exhaustively surveyed in vivo. NKG2D is known to be internalized upon ligand engagement (Lanier, 2015), so we reasoned that if NKG2D ligands are expressed and interact with NKG2D in healthy WT mice, antibody blockade of the relevant ligand(s) should result in increased levels of NKG2D on the surface of NK cells. Adult C57BL/6 (B6) mice were injected with confirmed blocking antibodies (Figure 1—figure supplement 1A) specific for NKG2D ligands RAE-1δ, RAE-1ε, or MULT1. NKG2D levels on NK cells were analyzed by flow cytometry 48 hr post-injection. In vivo blockade of RAE-1ε, but not RAE-1δ or MULT1, substantially increased NKG2D surface levels on NK cells in blood (Figure 1A), lymph nodes, and spleen (Figure 1—figure supplement 1B). NKG2D elevation after RAE-1ε blockade occurred as early as 12 hr after antibody injection (Figure 1B). We subsequently analyzed NKG2D surface levels in RAE-1-KO mice, which contain frameshift mutations (induced by CRISPR/Cas9) in the genes for both RAE-1ε and RAE-1δ (Deng et al., 2015). In healthy, unmanipulated animals, NK cells in RAE-1-KO mice showed substantially higher cell surface NKG2D levels than WT controls in all compartments tested, including blood, spleen, lymph nodes, and peritoneal wash (Figure 1C). NK cells in bone marrow and liver also showed elevated NKG2D levels in RAE-1-KO mice (Figure 1—figure supplement 2A). mRNA levels for Klrk1 (the gene for NKG2D) were identical in NK cells from WT and RAE-1-KO mice (Figure 1D), consistent with the conclusion that host RAE-1ε causes internalization of NKG2D from the NK cell surface. Blocking RAE-1ε in WT mice increased NKG2D to levels comparable to RAE-1-KO mice at steady state, whereas anti-RAE-1ε had no effect on NKG2D levels in RAE-1-KO mice (Figure 1—figure supplement 1C). Furthermore, blockade of RAE-1ε in combination with RAE-1δ in WT mice showed no additional effect on NKG2D levels compared with blocking RAE-1ε alone (Figure 1—figure supplement 1D). Figure 1 with 2 supplements see all Download asset Open asset NKG2D is engaged and internalized by constitutive interactions with endogenous RAE-1ε in vivo. (A) NKG2D surface levels measured by flow cytometry of blood NK cells 48 hr after injection of blocking antibody specific for the indicated NKG2D ligand. Data are representative of >4 independent experiments. (B) NKG2D surface levels on blood NK cells analyzed at the indicated time point after injection of anti-RAE-1ε. Data are representative of two independent experiments. (C) NKG2D surface levels on blood, lymph node, spleen, and peritoneal wash NK cells in RAE-1-KO mice or WT controls at steady state. Data are representative of >4 independent experiments. (D) Relative Klrk1 mRNA levels in blood NK cells sorted from WT or RAE-1-KO mice (n = 3) as measured by qRT-PCR. Data are representative of two independent experiments. (E) NKG2D surface levels on CFSE-labeled blood NK cells 48 hr after splenocyte transfer between WT and RAE-1-KO mice. Data are representative of two independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni post-tests (A, E) or a two-tailed unpaired Student’s t tests (C). Data represent means ± SEM. https://doi.org/10.7554/eLife.30881.003 To assess whether these phenotypes were intrinsic to NK cells, we transferred CFSE-labeled splenocytes from WT into RAE-1-KO mice and vice versa. When splenocytes were transferred from WT to RAE-1-KO mice, NKG2D levels on the transferred NK cells increased to match the RAE-1-KO mice (Figure 1E). Reciprocally, NKG2D surface levels were reduced on NK cells transferred from RAE-1-KO into WT mice. Cumulatively, these data demonstrated that in healthy WT mice a subset of cells express RAE-1ε, which engages and downregulates NKG2D at steady state from the surface of NK cells. Endogenous RAE-1ε diminishes NK responsiveness We next sought to understand the effect of host RAE-1ε on the function of NK cells. Splenic NK cell numbers and expression of CD11b and CD27 – cell surface markers associated with NK maturation (Hayakawa and Smyth, 2006) – were similar in WT and RAE-1-KO mice (Figure 2—figure supplement 1A). Release of cytotoxic granules and IFNγ are important NK cell functions (Vivier et al., 2008), so we analyzed these responses in WT and RAE-1-KO NK cells after acute ex vivo activation through a variety of receptors. We used a standard 5 hr responsiveness assay in which cells were stimulated by plate-bound antibodies that crosslink activating NK receptors, followed by flow cytometry for degranulation (marked by CD107a cell surface presentation) and intracellular IFNγ (Joncker et al., 2009, 2010). As is typical with this assay, stimulation through the activating receptor NKp46 triggered robust NK cell degranulation and IFNγ production from WT splenic NK cells, and a significantly greater percentage of NK cells from RAE-1-KO mice responded to stimulation compared with WT NK cells (Figure 2A and B). NK cells from RAE-1-KO mice also showed elevated responses when stimulated with platebound antibodies that ligate a distinct activating receptor, NK1.1, or that ligate NKG2D itself (Figure 2B). These data indicated that splenic NK cells from RAE-1-KO mice exhibit a hyper-responsive phenotype upon acute stimulation through a variety of activating receptors. Figure 2 with 1 supplement see all Download asset Open asset Endogenous RAE-1ε negatively regulates NK responsiveness. (A) WT or RAE-1-KO splenic NK cell IFNγ production and degranulation (CD107a) after 5 hr ex vivo stimulation with platebound control Ig or anti-NKp46. (B and C) Percentage of activated (IFNγ- and CD107a-double-positive) splenic or peritoneal NK cells from WT or RAE-1-KO mice after ex vivo stimulation with the indicated plate-bound antibody. Data are representative of >4 independent experiments. (D) Percentage of activated peritoneal NK cells after ex vivo stimulation from mice given control Ig or anti-RAE-1ε for the indicated time. Data are representative of two independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t tests. Data represent means ± SEM. https://doi.org/10.7554/eLife.30881.006 In our experience, NK cells in the peritoneal cavity typically yield relatively low responses to ex vivo stimulation. We tested whether endogenous RAE-1ε regulated the responsiveness of these cells. Interestingly, peritoneal NK cells from RAE-1-KO mice showed markedly greater responses compared with their WT counterparts when stimulated through NKp46, NK1.1 or NKG2D (Figure 2C). This especially large increase gave us a greater window to examine the desensitization effect, so we next analyzed peritoneal NK responses after injecting WT mice i.p. with antibodies that block RAE-1ε. Similar to the RAE-1-KO mice, blockade of RAE-1ε caused a substantial increase in NK responses to stimulation through all receptors tested (Figure 2D). The increased responses could be seen as early as 48 hr after antibody administration. To analyze killing of tumor cells, we performed a standard 4 hr 51Cr in vitro cytotoxicity assay, using YAC-1 cells as targets. Peritoneal wash cells from WT, RAE-1-KO, and NKG2D-KO mice were used as effectors. NKG2D-KO effectors were significantly less efficient at killing YAC-1 cells (Figure 2—figure supplement 1B), consistent with published reports showing that NKG2D-mediated recognition is required for efficient YAC-1 killing (Jamieson et al., 2002; Guerra et al., 2008). In contrast, RAE-1-KO mice showed markedly enhanced NK killing of YAC-1 cells (Figure 2—figure supplement 1B). Together, these data suggested that endogenous, steady-state RAE-1ε expression desensitizes NK responses to activation through multiple activating receptors and YAC-1 cells in vitro. NKG2D regulates NK responsiveness in a cell-intrinsic manner RAE-1ε binds NKG2D, so we expected NKG2D-KO NK cells to be hyper-responsive to NKG2D-independent stimuli. Indeed, NKG2D-KO NK cells from spleen and peritoneal wash showed increased responses to stimulation compared with WT controls when stimulated through NKp46 and NK1.1 (Figure 3A and B), as has also been previously reported (Zafirova et al., 2009; Sheppard et al., 2013; Deng et al., 2015). We then directly compared the responses of peritoneal NK cells from matched WT, RAE-1-KO, and NKG2D-KO mice. When stimulated with platebound antibody ligating NKG2D, NK cells from RAE-1-KO showed elevated responses, whereas NKG2D-KO NK cells failed to respond, as expected (Figure 3—figure supplement 1A). In contrast, stimulation through NKp46 resulted in elevated responses from both the RAE-1-KO and NKG2D-KO cohorts (Figure 3C). Interestingly, NKG2D-KO NK cells were consistently even more responsive than the NK cells from RAE-1-KO mice (Figure 3C). These data suggested that, in addition to RAE-1ε, other ligands may participate in NKG2D-mediated desensitization, or NKG2D may regulate NK responses partly through a ligand-independent mechanism in addition to the RAE-1ε-dependent mechanism documented herein. Figure 3 with 1 supplement see all Download asset Open asset RAE-1ε contributes to cell-intrinsic NKG2D-mediated regulation of NK responsiveness. (A and B) Percentage of activated splenic or peritoneal NK cells from WT or NKG2D-KO mice after ex vivo stimulation. Data are representative of >4 independent experiments. (C) NK activation in WT, RAE-1-KO, and NKG2D-KO peritoneal cells after ex vivo stimulation. Data are representative of three independent experiments. (D) NK activation after ex vivo stimulation of peritoneal cells from WT mice 8 weeks after lethal irradiation (11 Gy rad split dose) and reconstitution with bone marrow cells from WT (CD45.1) or NKG2D-KO (CD45.2) mice or a 1:1 mix. Data are representative of two independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni post-tests (C, D) or two-tailed unpaired Student’s t tests (A, B). Data represent means ± SEM. https://doi.org/10.7554/eLife.30881.008 We considered that NKG2D-mediated desensitization could happen in a cell-intrinsic manner – that is, through a given NK cell’s interaction with ligand and consequent desensitization – or cell-extrinsically via a specific population of ‘suppressor’ cells. To discriminate between these hypotheses, we generated bone marrow chimeras containing NKG2D-WT and NKG2D-KO cells in the same animal, or singly reconstituted chimeras as controls. WT (CD45.1) mice were lethally irradiated and reconstituted with bone marrow cells from WT (CD45.1) mice, NKG2D-KO (CD45.2) mice, or a 1:1 mixture of the two genotypes. Reconstitution efficiency was consistently greater than 99%, and the mixed chimeric mice contained similar numbers of WT and NKG2D-KO NK cells (Figure 3—figure supplement 1B). We then tested the chimeras for NK cell responsiveness. Consistent with our earlier data, NK cells from mice reconstituted with NKG2D-KO bone marrow showed greater responses than NK cells from mice reconstituted with WT bone marrow. Interestingly, NK cells from the mixed chimeras recapitulated these responses, as NKG2D-KO NK cells were hyper-responsive compared with WT NK cells in the same animals (Figure 3D). These data demonstrated that NKG2D desensitizes NK responses in a cell-intrinsic manner. Endothelial cells in lymph nodes as the primary source of endogenous RAE-1ε We next sought to identify the cellular source of RAE-1ε responsible for engaging NKG2D and desensitizing NK cells. We used a bone marrow chimera approach to restrict RAE-1ε expression to hematopoietic or nonhematopoietic cells. We used a radiation dose (600 Gy + 500 Gy split dose) that reliably led to replacement of >99% of cells in the hematopoietic compartment, although we cannot exclude the presence of some radio-resistant bone-marrow-derived cells in the chimeras. After irradiation, WT or RAE-1-KO mice were reconstituted with bone marrow from WT or RAE-1-KO mice, and NKG2D cell surface levels were analyzed on NK cells 8 weeks after reconstitution. As expected, KO → KO chimeras showed substantially higher NKG2D levels compared with WT → WT controls (Figure 4A) (Figure 4-figure supplement 1A). Chimeric mice in which RAE-1ε was present only in hematopoietic cells (WT → KO) showed high NKG2D levels comparable to KO → KO chimeras, indicating that hematopoietic RAE-1ε does not play a major role in engaging NKG2D, although was a effect in most experiments that failed to In contrast, mice with RAE-1ε expression to nonhematopoietic cells → recapitulated the low NKG2D levels seen in WT → WT animals (Figure 4A) (Figure supplement 1A). When we analyzed the responses of NK cells in these chimeras, a similar with nonhematopoietic RAE-1 a role in the desensitization of NK responses, although hematopoietic RAE-1 show some effect (Figure These data suggested that nonhematopoietic cells are the source of RAE-1ε that engages NKG2D and regulates NK cell responsiveness. Figure 4 with 2 supplements see all Download asset Open asset node endothelial cells as the endogenous source of RAE-1ε. (A) NKG2D cell surface levels on blood NK cells 8 weeks after WT or RAE-1-KO mice were lethally irradiated and reconstituted with WT or RAE-1-KO bone marrow. Data are representative of three independent experiments. (B) Percentage of activated NK cells from peritoneal cells from WT and RAE-1-KO bone marrow chimeras after plate-bound antibody stimulation. Data are representative of two independent experiments. (C) RAE-1ε expression on the indicated cell in lymph nodes from WT mice. Data are representative of >4 independent experiments. (D) RAE-1ε expression on endothelial cells in the indicated Data are representative of three independent experiments. Statistical significance was determined using one-way ANOVA and Bonferroni Data represent means ± SEM. We then a search for the nonhematopoietic source of RAE-1ε. RAE-1-KO mice had elevated NKG2D levels on NK cells in blood and other we reasoned that the cellular source of RAE-1ε must be to these NK cells as part of their normal we used flow cytometry to analyze RAE-1ε on nonhematopoietic cells in organs by NK cells. other NK cells to and from blood and nodes are for and have After of lymph nodes, of nonhematopoietic lymph node cells can be by expression of the molecule and the (Figure supplement et al., 2010). that are are blood endothelial cells and cells are endothelial cells engage these endothelial cells to enter and lymph nodes et al., cells are cells which a cellular that the lymph node et al., 2010). The population is We lymph nodes from mice and used flow cytometry to analyze RAE-1ε on these cells and showed little to no RAE-1ε, we found substantial RAE-1ε expression on and (Figure This was not to binding of the RAE-1ε because the in RAE-1-KO mice (Figure supplement 1C). we whether RAE-1ε was expressed on endothelial cells in other Splenic endothelial cells express low of RAE-1ε, but endothelial cells in the and showed little to no RAE-1ε (Figure and Figure supplement all other nonhematopoietic cells in these cell were also for RAE-1ε Endothelial endothelial cells are a subset of that into lymph nodes et al., cells can be using the antibody which recognizes a specific (Figure supplement et al., Interestingly, RAE-1ε expression was substantially higher on cells than the expression on (Figure supplement 1E). In these experiments showed that nonhematopoietic cells are the responsible for steady state NKG2D engagement and NK desensitization, and our of cellular RAE-1ε expression endothelial cells in tissue as the relevant cellular source for RAE-1ε. These findings suggest a in which NK cells, in and out of tissue are engaged and desensitized by RAE-1ε expressed on endothelial cells. Endothelial RAE-1ε and NKG2D engagement in the tumor NK cell responsiveness is by interactions and at of such as the tumor (Joncker et al., 2010; et al., 2014). The powerful antitumor of NK cells often for tumor cells and that can the NK response et al., 2014). To study the of endogenous RAE-1ε in tumor we used the called of a tumor that is to NK killing but NKG2D ligand expression et al., WT mice were with cells. After of tumors, mice were with antibodies RAE-1δ, RAE-1ε, or both for 48 after which the tumors were to and NK cells the tumors were analyzed for surface NKG2D levels. Blocking RAE-1ε but not RAE-1δ caused NKG2D on tumor-infiltrating NK cells (Figure tumors expression of NKG2D ligands, we an endogenous source of RAE-1ε, so we analyzed NK cells tumors in WT or RAE-1-KO mice. NK cells mice had elevated NKG2D in RAE-1-KO mice compared with WT controls (Figure Similar were when tumor-infiltrating NK cells were in mice with cells, which also NKG2D ligand expression (Figure Figure 5 with 1 supplement see all Download asset Open asset Endothelial RAE-1ε and NKG2D engagement in the tumor (A)