HHS Public Access Author manuscript Author Manuscript
Immunity. Author manuscript; available in PMC 2017 August 16. Published in final edited form as: Immunity. 2016 August 16; 45(2): 280–291. doi:10.1016/j.immuni.2016.07.005.
Cell-extrinsic MHC class I molecule engagement augments human NK cell education programmed by cell-intrinsic MHC class I Jeanette E. Boudreau1, Xiao-Rong Liu1, Zeguo Zhao1, Aaron Zhang1, Leonard D. Shultz2, Dale L. Greiner3, Bo Dupont1,4, and Katharine C. Hsu1,4,5,*
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1Immunology
Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA 2The
Jackson Laboratory, Bar Harbor, ME 04609
3Program
in Molecular Medicine Diabetes Center of Excellence, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655 4Department 5Weill
of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
Cornell Medical College, New York, NY, 10065, USA
Summary Author Manuscript
The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by “self” MHC class I molecules in a process called “education”. In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B* 27:05+ transgenic mice and patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34+ stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1+ NK cells gained reactivity after adoptive transfer to HLA-B*27:05+ mice or bone marrow chimeric mice where HLA-B*27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell–intrinsic HLA in maintaining NK cell education.
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eTOC Blurb Killer immunoglobulin-like receptors (KIR) and human leukocyte antigens (HLA) calibrate NK cell education. Hsu and colleagues demonstrate that NK cell reactivity increased or decreased,
*
Corresponding author: Dr. Katharine C. Hsu, Department of Medicine, Memorial Sloan Kettering Cancer Center, [email protected]. Author contributions L.D.S. and D.L.G. established the B27 Tg+ mice; J.E.B., X-R.L., A.Z., Z.Z., B.D. and K.C.H. designed and performed experimental analysis; J.E.B., B.D. and K.C.H. wrote and edited the manuscript.
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respectively, when HLA was acquired from neighboring cells or was silenced in educated cells. Therefore, both cell-intrinsic and –extrinsic HLA co-operate to maintain and adjust NK cell education.
Author Manuscript Introduction
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Natural killer (NK) cells integrate activating and inhibitory signals to measure the health of neighboring cells. The major histocompatibility complex (MHC) class I antigens, termed human leukocyte antigens (HLA) in humans are recognized by NK cells as markers of “healthy self” via inhibitory Ly49 receptors in mice or killer immunoglobulin-like receptors (KIR) humans, respectively. Downregulation or loss of class I expression resulting from infection, damage, or transformation, interrupts inhibition and triggers NK cell reactivity through simultaneous activating signals (Long et al., 2013). The capacity of NK cells to recognize cells lacking MHC class I is well established, but the processes by which NK cells are calibrated to recognize “self” MHC class I molecules are not.
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Individual NK cells exhibit variable expression of inhibitory receptors; consequently, NK cells exhibit different sensitivity to inhibition based on the receptor(s) they express and the co-inherited MHC class I (Raulet et al., 2001). A process called “education” calibrates NK cells to recognize and respond to cells that have lost expression of MHC class I (Anfossi et al., 2006; Joncker et al., 2009; Kim et al., 2005). NK cells that express KIR that are specific for “self” MHC class I exhibit greater responsiveness against MHC class I− targets than NK cells that lack inhibitory receptors for “self” MHC class I. Paradoxically, the same interactions between self-specific KIR and MHC class I that program education also govern NK inhibition when the putative target cell expresses the self-MHC class I ligand. Altogether, the reactivity of NK cells is balanced to permit sensitivity to damaged cells without autoimmune consequences. The processes governing NK cell education are not fully understood. In the “licensing” model, NK cells are endowed with functional potential when their inhibitory receptors can Immunity. Author manuscript; available in PMC 2017 August 16.
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engage “self” MHC class I (Anfossi et al., 2006; Kim et al., 2005). In another model, NK cells that lack inhibitory receptors sensitive to “self” MHC class I may be “disarmed” via a process analogous to activation-induced anergy. The capacity for inhibition by “self” MHC class I protects NK cells from anergy, enabling educated NK cells to retain functional capacity (Raulet, 2006).
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The genes for KIR and Ly49 segregate independently from those of MHC class I, and the expression of inhibitory receptors varies among NK cells in an individual’s repertoire (Parham, 2005). As a result, the education of each NK cell is calibrated by its receptor expression and the availability of cognate MHC class I molecules. In mice, NK cell-derived Ly49 molecules interact with cognate MHC class I molecules on both neighboring cells (trans) and on the NK cell itself (cis) (Doucey et al., 2004; Ioannidis et al., 2001; Scarpellino et al., 2007). Via a flexible stalk, Ly49 bends to make the same binding site available to MHC class I ligands in trans and cis (Back et al., 2009; Chalifour et al., 2009; Tormo et al., 1999). Mutation of the stalk to allow trans but not cis binding diminishes NK cell responses to MHC− targets, leading investigators to conclude that Ly49 interactions in cis with MHC class I molecules contribute to mouse NK cell reactivity (Chalifour et al., 2009). Supporting an additional role for trans ligands in mouse NK cell education, mature NK cells adoptively transferred from beta-2 microglobulin (β2m)-deficient mice to MHC class I-sufficient WT hosts results in an increase in NK cell responsiveness (Elliott et al., 2010; Joncker et al., 2010). Reciprocally, NK cells transferred from WT mice to β2m-deficient hosts exhibit decreased reactivity (Joncker et al., 2010). Mixed chimeras of MHC class I moleculesufficient and -deficient stem cells reveal that MHC class I molecules on either hematopoietic or stromal cells can educate mouse NK cells expressing cognate receptor, and responsiveness is highest when MHC class I molecules are present in both compartments (Xie et al., 2010). Together, these findings illustrate that MHC class I molecules expressed by both NK and neighboring cells contribute to NK cell education in mice.
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Although the Ly49 and KIR proteins both educate NK cells, they are the products of distinct non-orthologous gene families (Parham, 2005). Consequently, it has been difficult to translate mouse findings to understand human NK cell biology. Taking cues from the known patterns of NK cell education in mice, we investigated the roles of cell-intrinsic and – extrinsic MHC class I molecules for educating human NK cells. Using a mouse transgenic for HLA-B*27:05, which exhibits the Bw4-ligand for KIR3DL1, we investigated the relative contributions to NK education by HLA class I molecules presented in trans from hematopoietic or stromal cells and present in the NK cell itself. While human NK cell reactivity was augmented by HLA class I available in trans, cell-intrinsic HLA class I was necessary to maintain education. Using RNA interference, we showed that HLA class I molecules on the NK cell itself were required to maintain human NK cell education. HLA class I molecules were readily adopted from trans sources by NK cells after cell transfer, a potential mechanism by which environmental HLA class I molecules can adjust human NK cell reactivity. In patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), we found that donor-derived NK cells analogously exhibit recipient HLA class I molecules on the cell surface, and education that reflects both donor and recipient HLA class I. Altogether, these findings illuminate important aspects in the biology and calibration of
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human NK cell function that may be informative in HCT and adoptive cell therapies, where HLA class I alleles in the donor and recipient may differ.
Results HLA-B*2705 Tg+ and Tg− mice support human NK cell development
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Immunocompromised and humanized mice are increasingly used to study human NK cell function in vivo (Chen et al., 2009; Huntington et al., 2009; Pek et al., 2011). However, these models have not been engineered to express human HLA class I molecules, limiting their utility in investigations of human NK cell development and education. We used a NOD.CgRag1−/−IL2rgtm1Wjl /Sz strain with transgenic expression of human B2M and the HLABw4+ HLA-B*27:05 (B27 Tg+) to understand how interactions with HLA class I molecules impact the education of human NK cells (Figure 1A) (Taurog et al., 1990). Both B27 Tg+ and their Tg− littermates (Tg−) supported the in vivo development of human lymphocytes from CD34+ hematopoietic stem cells, achieving full human chimerism in blood lymphocytes by 7 weeks post-transplant (Figure 1B). Semi-weekly injections of BA/F3 feeder cells that trans-present IL-15 (Pittari et al., 2013) favored human NK cell development in both strains. A greater number of NK cells were recovered from the spleens of B27 Tg+ mice at week nine (Figure 1C) and until at least week 11 (data not shown), suggesting that the presence of HLA class I molecules in trans supports enhanced survival and/or proliferation of NK cells. Humanized mice additionally exhibited monocytes, B and T cells of human origin (Supplemental Table 1).
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NK cells developing from CD34+ stem cells in the humanized mice expressed phenotypic markers similarly to NK cells from the peripheral blood of healthy human donors, with the exception of NKG2A, which was expressed on a majority of NK cells in the humanized mice (Figure 1D–E). CD16, NKG2D and NKp46 were all detectable on the surface of NK cells harvested from the blood and spleen and did not differ between B27 Tg+ and Tg− mice (Figure 1F–I). Inhibitory KIR were expressed on a greater percentage of NK cells developing in Tg− compared with B27 Tg+ mice (Figure 1J–L), consistent with previous studies that demonstrate overexpression of inhibitory receptors in the absence of their ligands (Van Bergen et al., 2013). Similarly, CD57 was expressed at higher frequency among NK cells in Tg− mice (Figure 1M). Human NK cells are educated by cognate HLA-Bw4 ligand in trans
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NK cell effector function is primed in vivo by inflammation, prompted by pathogenassociated molecular patterns (PAMPs) and/or cytokines (Boudreau et al., 2011; Lucas et al., 2007; Strowig et al., 2010). Similar to other humanized mouse models, priming with LPS was necessary for an NK cell response to stimulation with IL-12+IL-18 or the MHC class I− target cell lines, 721.221 or K562; NK cells from unprimed, donor-matched mice were not responsive (Ito et al., 2002; Tanaka et al., 2012) (Supplemental Figure 1). To study NK cell education, we analyzed CD34+ stem cell donors exhibiting cell-surface KIR3DL1, the cognate receptor for HLA-Bw4 (Gumperz et al., 1995). NK cells developed in the humanized mice, including KIR3DL1+ NK cells, degranulated robustly in response to
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K562 cell line targets similar to healthy human donors (Figure 2A). When pairs of Tg− and B27 Tg+ mice were transplanted with CD34+ cells from the same donors, NK cells developing in B27 Tg+ compared with Tg− mice exhibited higher degranulation (Figure 2B). NK cells exclusively expressing KIR3DL1, and therefore lacking KIR2D receptors, exhibited elevated responsiveness (Figure 2C), suggesting that availability of the HLA-Bw4 ligand in B27 Tg+ mice can promote education of developing NK cells expressing the cognate receptor. IFN-γ production was similarly elevated among KIR3DL1+ NK cells from B27 Tg+ mice compared with Tg− animals (Supplemental Figure 2A–B). Donor and recipient HLA class I molecules are required for maximum reactivity
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Reflecting variability in NK cell responses observed between individual humans (Yawata et al., 2006), substantial inter-donor variation in KIR3DL1+ NK cell responsiveness of humanized mice was evident (Figure 2C). If HLA class I molecules contributed by the mouse stromal environment solely determined NK cell education, such variability would not be predicted to occur among our inbred mice. We therefore considered the possibility that the HLA class I molecules from the human stem cell donor might also contribute to NK cell education. To distinguish the educating impact of HLA class I molecules presented by neighboring human hematopoietic cells from HLA class I molecules presented by mouse stromal cells, we examined the cytotoxic and cytokine responsiveness of KIR3DL1+ NK cells that developed in B27 Tg+ or Tg− mice, stratified by the presence or absence of Bw4 in the human CD34+ stem cell donor (Figure 2D, Supplemental Figure 2C). The most responsive cells were KIR3DL1+ NK cells derived from Bw4+ donors and developed in B27 Tg+ mice. KIR3DL1+ NK cells derived from Bw4+ donors and developed in Tg− mice and NK cells derived from Bw4− donors and developed in B27 Tg+ mice exhibited similar responsiveness. The least responsive cells were KIR3DL1+ NK cells derived from Bw4− donors and developed in Tg− hosts. The stepwise escalation of NK cell responsiveness indicates that both donor and host HLA class I molecules influenced NK cell education.
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A contribution of hematopoietic HLA class I molecules to NK cell education was further supported by examination of the KIR3DL1− cells, which demonstrated responsiveness in both Tg− and B27 Tg+ hosts (Figure 2A and D). If HLA class I molecule presentation by mouse stromal cells was solely responsible for NK cell education, we would expect that NK cells expressing KIRs other than KIR3DL1 would be uneducated and hyporesponsive. Therefore, we compared the responsiveness of NK cells expressing KIR2DL2 or KIR2DL3, but lacking other inhibitory KIR, based on the presence or absence of cognate HLA-C group 1 (HLA-C1) ligand in the CD34+ stem cell donor. KIR2DL2+ or KIR2DL3+ NK cells from HLA-C1+ donors developed in either B27 Tg+ or Tg− mice exhibited greater responsiveness compared to KIR2DL2+ or KIR2DL3+ NK cells from HLA-C1− donors (Figure 2E). We conclude that HLA class I molecules presented by donor hematopoietic cells impacts NK cell education. Donor and recipient HLA class I molecules contribute to NK cell education in patients following hematopoietic cell transplantation To understand if our findings in humanized mice reflect human biology, we examined NK cell education in recipients of HLA mismatched hematopoietic cell transplantation (HCT).
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We compared NK cells exhibiting KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 as their sole receptor, stratifying based on the presence or absence of their ligands in the HCT donor and/or transplant recipient. Analogous to the findings in the mouse model, HLA-Bw4 contributed by both the stem cell allograft and the recipient stromal compartment promoted KIR3DL1+ NK cell responsiveness to K562 cells: the highest responsiveness occurred when HLA-Bw4 was available in both (Figure 3A). The educating ligands for KIR2DL1 (HLAC2) and KIR2DL2 or KIR2DL3 (HLA-C1), contributed similarly to NK cell education: ligand present in both the allograft and recipient was associated with the highest reactivity, and similar NK cell responsiveness occurred when the KIR ligand was present in either the stem cell allograft or the recipient (Figure 3B and C). HLA-Bw4 in trans re-educates mature NK cells
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To determine whether NK cell education is a process that persists after egress from the bone marrow, we transferred mature peripheral blood NK cells from the same human donors into pairs of B27 Tg+ or Tg− mice. Human NK cells were detected in the peripheral blood, lymphoid and peripheral tissues for at least 2 weeks after transfer (Figure 4A), and exhibited similar proliferation in B27 Tg+ and Tg− mice (Figure 4B). After two weeks in B27 Tg+ mice, KIR3DL1+ cells exhibited enhanced responsiveness compared with the same NK cells pre-transfer or those incubated in Tg− mice (Figure 4C). Notably, degranulation of mature KIR3DL1+ NK cells did not decrease in an environment lacking HLA-Bw4, indicating that NK cell education was maintained even in the absence of environmental ligand. Gain of function was most readily observed among cells from Bw4− donors transferred to the B27 Tg+ mouse, demonstrating that uneducated NK cells can modulate their education despite being fully mature (Figure 4D). Similarly, KIR3DL1+ NK cells adoptively transferred to B27 Tg+ mice exhibited enhanced IFN-γ production compared with NK cells from the same donor, but transferred to Tg− animals (Supplemental Figure 3). To ascertain if the increased reactivity of NK cells by environmental HLA-Bw4 was specific to KIR3DL1+ cells rather than a general consequence of an altered MHC class I environment, we assessed the reactivity of NK cells solely expressing KIR2DL1, KIR2DL2 or KIR2DL3, comparing the difference in reactivity after transfer from the same donor to B27 Tg+ or Tg− mice (Figure 4E). While KIR3DL1+ NK cells exhibited a 2.5-fold higher increase in effector function in B27 Tg+ mice compared with Tg− mice, KIR2DL1+ and KIR2DL2+ or KIR2DL3+ NK cells had no change in response from pre-transfer (data not shown), and there was no difference in response of KIR2DL1+, KIR2DL2+ or KIR2DL3+ NK cells between mouse strains. Therefore, the upward tuning of NK cell reactivity required cognate HLA class I molecules (Figure 4E and Supplemental Figure 3).
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Gain of function in NK cells was mediated by stromal and hematopoietic cells To better understand the cellular interactions that facilitate the increased function of NK cells by local ligands, we compartmentalized HLA-Bw4 to hematopoietic or stromal cells by creating bone marrow chimeric mice (Figure 5A). Lethally irradiated B27 Tg+ or Tg− mice were reconstituted with whole bone marrow derived from either strain to localize HLA-Bw4 to the donor (D) or recipient (R) compartments (D+R− or D−R+). Tg− mice reconstituted with Tg− bone marrow (D−R−) and B27 Tg+ mice reconstituted with B27 Tg+ bone marrow
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(D+R+) served as controls. After nine weeks, mice had a fully reconstituted hematopoietic compartment. NK cells from healthy human donors were then transferred into these mice to determine whether the hematopoietic (donor), stromal (recipient), or both compartments were responsible for increasing NK cell function. The responsiveness of NK cells derived from the same human donors was thereafter compared in quartets comprised of each combination of HLA-Bw4 localization. Whether HLA-Bw4 was confined to the hematopoietic or stromal cells or present in both, KIR3DL1+ NK cells demonstrated enhanced responsiveness compared to NK cells from the original donor and to NK cells transferred to mice lacking HLA-Bw4 (Figure 5B–C). The responsiveness of KIR2DL2+ or KIR2DL3+ cells from the same donor was not significantly altered in different chimeric constructions (data not shown). We conclude that any trans source of cognate ligands for KIR, hematopoietic or stromal, was sufficient to increase the reactivity of mature NK cells.
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HLA class I molecules are required on NK cells for education
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The observation that NK cell responsiveness persists in the absence of environmental HLA class I after adoptive transfer suggests that maintenance of NK cell education does not require persistent interaction with ligand in trans. We hypothesized that cognate MHC class I molecules on the NK cell are necessary for their function and tested this by introducing shRNA via lentiviral infection to silence expression of β2m in primary NK cells (Figure 6A). Gating for KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 monopositive cells enabled comparison of NK cell function in the presence or absence of normal HLA class I molecule expression. Among each of the KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 monopositive populations, knockdown of β2m induced a significant reduction of responsiveness against K562 cells (Figure 6B). This loss of function was especially profound among educated NK cells. In a representative donor expressing HLA-C1 and HLA-Bw4, β2m silencing diminished the reactivity of educated NK cells expressing KIR2DL2, KIR2DL3 or KIR3DL1 (Figure 6C). After β2m silencing, the reactivity of these NK cells was similar to that of the uneducated KIR2DL1+ population, which was unaffected by loss of β2m. Among 34 different donors, β2m silencing significantly impacted the responsiveness of the educated NK cell populations, where loss of “self” HLA class I molecules led to a 50–60% loss in reactivity (Figure 6D). Mature NK cells can acquire HLA class I molecules from trans sources
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Our findings indicate that HLA class I molecule ligands present in cis maintain NK cell reactivity and in trans augments NK cell reactivity. We hypothesized that trans ligands could be acquired by NK cells. We labeled NK cells from HLA-Bw4− donors with CellTrace Violet (CTV) and measured HLA-Bw4 staining on their membranes after their transfer to B27 Tg+ mice. Within two days of transfer, mouse CD45 was evident on the majority of CTV+ human cells harvested from the blood and organs of both B27 Tg+ and Tg− mice (Figure 7A–B). CTV+ NK cells additionally displayed HLA-Bw4 after incubation in B27 Tg+ but not Tg− mice, demonstrating that NK cells could acquire HLA class I molecules from the recipient (Figure 7C). A similar rate of mouse CD45 uptake was observed among CTV+ cells transferred to both mouse strains (Figure 7D), and HLA-Bw4 uptake was not restricted to KIR3DL1+ NK cells (data not shown). We therefore conclude that cell surface
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protein transfer occurred as a likely consequence of cell-cell interaction, rather than a process mediated by specific receptor-ligand engagement. To determine if HLA-Bw4 acquired from trans sources were associated with gain of function among NK cells, we compared the reactivity of KIR3DL1+ NK cells with or without uptake of HLA-Bw4. CTV+ NK cells isolated from HLA-Bw4− donors staining positive for HLA-Bw4 following transfer to B27 Tg+ mice exhibited enhanced responsiveness compared with HLA-Bw4− NK cells from the same mice (Figure 7E). In contrast, CTV+ NK cells from the same donors transferred to Tg− mice did not exhibit HLABw4 staining or enhanced responsiveness (Figure 7D–E).
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To confirm that these findings were not an artifact of our xenograft model, we examined lymphocytes collected from recipients of HLA-mismatched HCT to discern whether recipient stromal HLA class I molecule might similarly be transferred to donor-derived NK cells in humans. NK cells from patients who had full donor chimerism at one year post-HCT were evaluated. Similar to the mouse studies, NK cells derived from an HLA-Bw4− stem cell donor exhibited HLA-Bw4 expression a year after developing in an HLA-Bw4+ patient (Figure 7F). Altogether, our data support a role for cell-intrinsic HLA class I molecules to maintain NK cell reactive potential and suggest that the requisite ligands might be acquired via membrane transfer from neighboring cells.
Discussion
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Using a humanized mouse transgenic for HLA-B*27:05, we have demonstrated that human NK cell education is shaped by HLA class I molecules present in and around NK cells, where cell-intrinsic HLA class I molecules maintain NK cell reactivity that can be augmented by cognate HLA class I molecules provided by neighboring cells. These findings extend to patients receiving KIR ligand-disparate HCT, in whom reconstituting donorderived NK cells exhibit education that reflects both the donor and recipient HLA class I molecules. KIR3DL1+ NK cells acquired higher responsiveness upon transfer to or following development in an environment replete with cognate HLA class I molecules. Removal of HLA class I molecules from the cell itself by shRNA could diminish the reactivity of an educated NK cell, underscoring a role for cell-intrinsic HLA class I molecules in the maintenance of NK cell education. We found that short interactions were sufficient to transfer HLA class I molecules between mouse stromal cells and transferred human NK cells, and we could identify uptake of recipient HLA class I molecules on donorderived NK cells in HCT patients. It is therefore plausible that trans HLA class I molecules may be recycled for cell-intrinsic interactions.
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Education of human NK cells can occur via HLA class I molecules available in the stromal, hematopoietic and NK cell compartments. For developing and mature NK cells, a source of trans ligand contributed to NK cell education, with the highest reactivity achieved when cognate ligand was available in both trans and cis. NK cell education and maturation were separable, because lack of trans ligand or education did not prevent NK cell differentiation in the Tg− mouse. Similarly, maturation did not prevent subsequent enhancement of NK cell reactivity after transfer of NK cells to B27 Tg+ mice. It is worth noting that the presence of
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cognate ligand in trans alone was insufficient to drive terminal maturation: priming was required before human NK cells developed in vivo were responsive toward MHC class I− target cells. The finding that human NK cells require priming before they exhibit functional education provides insight into human NK cell biology, where testing for the efficacy of unprimed NK cells has not previously been possible. In contrast to observations in mice, we find that educated mature NK cells do not lose responsiveness upon transfer to an environment devoid of HLA class I molecules (Elliott et al., 2010). This finding likely reflects a fundamental distinction in mouse and human NK cell biology that is pronounced in HLA class I-mismatched HCT in patients, where we have demonstrated that NK cell education retained the imprint of donor and recipient HLA class I molecules even a year after HCT.
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Our data reveal that while NK cells could be educated by trans ligands, cell-intrinsic HLA class I molecules played a critical role in the maintenance of NK cell effector potential, a finding supported by the functional consequences of silencing cell-intrinsic HLA class I molecules. The contributions of cell-intrinsic and -extrinsic HLA class I were not mutually exclusive: membrane proteins, including HLA class I, were acquired by adoptively transferred NK cells, leading to acquisition of cis KIR ligands from trans compartments and reflecting a similar process that occurs in mice (Miner et al., 2015). The transfer of membrane proteins is known to occur non-specifically between NK cells and neighboring cells (Carlin et al., 2001; Chow et al., 2013). Contrary to findings in mice, where Ly49 can facilitate uptake of specific MHC ligands (Sjöström et al., 2001), our findings do not support a receptor-specific acquisition of HLA-B*27:05.
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Analogous to mice, where cis interactions between Ly49 and MHC facilitate NK cell education (Bessoles et al., 2013; Chalifour et al., 2009; Doucey et al., 2004; Held and Mariuzza, 2008), cis interactions between KIR and HLA class I molecules may also program NK cell reactivity. Trans ligands may be relevant to NK cell education in at least two ways: by providing a source of HLA class I molecules which, when acquired by NK cells, can be recycled for cis interactions; and/or by providing an inhibitory signal to rescue NK cells from “disarming” via chronic activation (Raulet, 2006). Whether the availability of ligands in trans is sufficient to re-educate NK cells or the acquisition of HLA class I molecules from neighboring cells merely marks NK cells which have engaged them remains to be determined.
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Ly49 and KIR are different molecules that evolved to fulfill remarkably similar roles: establishment of NK cell reactivity and maintenance of self-tolerance. Both molecules are rapidly evolving, and both trigger conserved signal transduction pathways (Kelley et al., 2005; Long et al., 2013). While Ly49 molecules possess a flexible stalk region, which permits cis and trans interactions with MHC via the same binding site, KIR molecules lack such a region, which has made a cis interaction between KIR and HLA class I molecules seem unlikely (Held and Mariuzza, 2008). HLA class I molecules are known to interact in cis with the LILRB1 receptors, however, establishing precedent for a cis interaction in human NK cells that does not rely on a flexible stalk (Li et al., 2013). It is feasible that cellintrinsic HLA class I molecules may bind KIR molecules in cis, to establish a mechanism of
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NK cell education analogous to Ly49 and MHC (Chalifour et al., 2009; Held and Mariuzza, 2008). Environmental changes, including alterations in HLA class I ligand availability for inhibitory receptors, might influence NK cell education. Adjustment would allow real-time re-calibration of effector function for sensitivity to the local environment (Pradeu et al., 2013). Such a mechanism may be beneficial in pregnancy, where upward re-setting of maternal decidual NK cell education by fetal antigens may facilitate trophoblast invasion (Sharkey et al., 2015). NK cell education may also impact outcomes in HCT or adoptive cell therapies, where HLA class I molecules may differ between juxtaposed donor and recipient cells.
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In HCT, gain of NK cell function is only beneficial to the patient. Reports of lower leukemia relapse rates following HLA class I-mismatched HCT may be due in part to enhanced NK cell anti-leukemic surveillance (Ruggeri et al., 2002). Because the risk of relapsed disease following HCT remains disappointingly high, with nearly always fatal consequences, our findings of NK cell education by recipient HLA class I without loss of NK cell education by donor HLA class I support the judicious use of HLA class I molecule-mismatched alternative donor sources. Consideration of HLA mismatched donors for HCT, regardless of haploidentical, umbilical, or unrelated origin, should include donor KIR genotyping to maximize NK cell reactivity.
Experimental procedures Mice
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The Tg(B2M, HLA-B*27:05)56-3Trg allele was generated by injection into the male pronucleus of (C57BL/6J x SJL) fertilized eggs (Taurog et al., 1990). This strain was imported into The Jackson Laboratory and backcrossed onto NOD/Lt until N9 and was then mated to maintain the transgene in the hemizygous state, as it is lethal in the homozygous state. NOD.Cg-Tg(B2M,HLA-B*27:05)56-37Tg/Dvs mice were mated with NOD.CgRag1tm1MomIL2rgtm1Wjl/Sz mice to create the NOD.Cg-Rag1tm1MomIL2rgtmWjl Tg(B2M, HLA-B* 27-05)56-3 mouse strain (B27 Tg+). Expression of B27 on mouse tissues was confirmed by FACS. NOD.Cg-Rag1tm1MomIL2rgtm1Wjl/Sz (NRG) littermate mice (Tg−) were used as controls in all experiments. Mice were maintained in specific pathogen-free conditions and bred in the MSKCC Research animal Resource Center, with amoxicillin and Baytril in food and water given ad libitum. Animal experimentation was conducted in specific pathogen-free conditions and approved by the MSKCC Institutional Animal Care and Use Committee (IACUC).
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Mouse blood and spleen PBMC collection Blood was collected in heparinized tubes by retro-orbital bleeding under isoflurane anesthesia. For timecourse experiments, matched trios of mice with the same transgenic background were engrafted with NK cells from the same donor and alternately sampled to allow a minimum 10-day rest period between bleedings.
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Mice were euthanized by cervical dislocation under isoflurane anesthesia. Spleens were collected in RPMI, dissociated by gentle mashing with a syringe plunger and filtered through 40 μM mesh (BD biosciences, San Jose, CA). Red blood cells were lysed using ammonium-chloride-potassium lysis buffer (ACK) (MSKCC core facilities). Purification and adoptive transfer of human NK cells Umbilical cord blood and buffy coats from healthy human donors were obtained from the New York Blood Center (nybloodcenter.org). Samples were anonymized prior to laboratory analysis; therefore, additional research consent requirements were waived by the MSKCC IRB. Additional PBMC collected from buffy coats, were cryopreserved prior to analysis and stimulated with 1000 IU IL-2/mL overnight prior to activation and FACS analysis.
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DNA was isolated from whole blood using the Qiagen Blood DNeasy kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). KIR typing was performed as described (Hsu et al., 2002; Vilches et al., 2007). A KIR ligand typing kit (Olerup, West Chester, PA) was used to identify individuals exhibiting HLA-Bw4, HLA-C group 1 (HLA-C1) or HLAC group 2 (HLA-C2). NK cells or CD34+ stem cells were isolated by Rosettesep negative selection (Stemcell Technologies, Vancouver, Canada) and confirmed to be >95% pure by FACS. NK cells were adoptively transferred to mice or cryopreserved prior to in vitro analysis. BA/F3 with human IL-15Rα and IL-15
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The mouse pre-B lymphocyte cell line BA/F3 was stably co-transfected with IL-3, IL-15 and IL-15Rα and selected by limiting dilution to create BA/F3 cells that transpresent IL-15 on the IL-15Rα stalk (Pittari et al., 2013). BA/F3-IL15 + IL15Rα stable transfectants were irradiated (100 cGy, cesium-137 irradiator) and 5 × 106 cells were injected intravenously twice weekly to support NK cells in vivo beginning three weeks after CD34+ transplant or immediately after adoptive transfer of NK cells. To prime NK cells derived in vivo, mice were injected with LPS (10 mg/mouse) unless otherwise indicated. Analysis of NK cell proliferation NK cell proliferation was quantitated by CellTrace violet (CTV) dilution (Life Technologies, Eugene, OR). Prior to injection, purified NK cells were labeled with 5 μM CTV. CTV dilution was quantitated in CD56+ cells by flow cytometry (LSR Fortessa, BD Biosciences) and analyzed with FlowJo 9.7.6 software (Ashland, OR). HCT patients and sampling
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All studies using samples from HCT patients were approved by the MSKCC IRB, and all patients consented to the research study. Peripheral blood lymphocytes from 12 patients receiving double-unit cord blood transplantation for the treatment of hematologic malignancies were collected 200–365 days after transplantation, a time at which a dominant donor cord blood unit has engrafted and NK cell reactivity is stabilized (Purtill et al., 2014; Yu et al., 2009). All studied patients were confirmed to have 100% donor chimerism provided by one cord source. KIR genotyping was performed on genomic DNA from cord blood, as described (Hsu et al., 2002). MHC class I allele typing was facilitated by the Immunity. Author manuscript; available in PMC 2017 August 16.
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National Marrow Donor Program, and alleles were assigned to KIR epitope groups using the Immunopolymorphism database (http://www.ebi.ac.uk/ipd/). Cryopreserved PBMC were thawed overnight in RPMI containing 10% FBS and 1000 IU/mL IL-2. PBMC were stimulated with K562 target cells at a 3:1 ratio for 5h and assessed for degranulation within NK cell populations expressing KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 as their sole inhibitory receptor. Stimulation and flow cytometry analysis
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The HLA class I− human target line for NK cells, K562, was grown from mycoplasmanegative stocks and maintained in RPMI containing 10% FBS. NK cells harvested from mice were stimulated using K562 cells (3:1 ratio of PBMC:K562) for 5h in the presence of anti-CD107a (H4A3, BD Biosciences). For measurement of IFN-γ production, 20 ng/mL BFA and 0.03% monensin were added during the final 3.5h of culture to inhibit protein export by NK cells. Non-specific antibody binding was blocked by addition of 1μg/mL total mouse IgG (Sigma-Aldrich, St. Louis, MO). Viable human NK cells were identified by excluding mouse CD45+ cells and dead cells, and gating for human CD56+ cells (live/dead fixable dye, ThermoFisher Scientific, Grand Island, NY). Representative staining and gating are shown in Supplemental Figure 4. All antibodies, clones and sources used to identify NK cell subsets are shown in Supplemental Table 2. Silencing of HLA class I expression
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HLA class I expression was targeted using shRNA against β2m, HLA-B or HLA-C (SigmaAldrich). Cocktails of shRNA were cotransfected with gag-pol and VSV glycoprotein into Phoenix A packaging cells in the presence of 10 μg/mL chloroquine (Naldini, 1998). Control viruses included CFP in lieu of shRNA. Viral supernatants were collected 48–72h posttransfection and transferred to primary NK cells in the presence of 5 μg/mL polybrene and spinfected at 300 × g for 90 min. NK cells were cultured for 48h in RPMI + 10% FBS and 300 IU/mL IL-2 prior to stimulation with K562 cells for functional assessment. Statistical analysis
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Paired samples analysis—To compensate for inter-donor variation and allow for comparison between experiments, paired samples representing the same donor after exposure in vivo to B27 Tg+ or Tg− environments were compared. The fold change in B27 Tg+ or Tg− (%CD107a+ KIR3DL1+ NK cells in B27 Tg+ mice / %CD107a+KIR3DL1+ NK cells in Tg−), or the fold change in in vivo compared with donor (%CD107a+KIR3DL1+ NK cells in B27 Tg+ mice / %CD107a+KIR3D1+ NK cells in donor) was calculated and is displayed in graphs, as indicated. Statistical analyses were completed using paired samples t tests.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments We thank Dr. Thomas A. Waldmann (National Cancer Institute) for cDNA clones for human IL-15 and human IL-15Rα. We thank Dr. Stuart Tangye (Garvan Institute, Darlinghurst, Australia) for providing the mouse pre-B cell line BA/F3, immortalized with mouse IL-3 gene. The authors thank Drs. Joseph Sun and Alan Hanash (Memorial Sloan Kettering Cancer Center) for helpful comments on the manuscript. We are grateful to Chi-Hang Cheung, Jean-Benoît Le Luduec, Shajoti Rahman, and Mimi Tang for technical assistance. This work was supported by NIH P01 (CA23766) and NIH R01 HL088134 to KCH; NIH NCI CA08747 and CA023766 to BD; core grant (CA034196) and NIH R24OOD018259 and UC4DK104218 to LDS and DLG and Core Grant (P30 CA008748) to MSKCC. Additional support was provided from the William H Goodwin and Alice T Goodwin and the Commonwealth Foundation for Cancer Research and the Helmsley Charitable Trust 2012PG-T1D018 (BD).
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Highlights 1.
Human NK cell education reflects cell-intrinsic and -extrinsic HLA class I
2.
NK cells can be educated by HLA class I from hematopoietic and stromal cells
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NK cell-intrinsic HLA class I maintains education via interaction with KIR
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NK cell function is enhanced after trans-acquisition of cognate HLA class I
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Figure 1. Development and characterization of an HLA class I-expressing humanized mouse model
(A) HLA-Bw4 expression on murine splenocytes. Dashed grey histogram represents B27 Tg+ mice, and the solid black histogram represents Tg− mice. (B) Engraftment of human cells from CD34+ stem cells in B27 Tg+ (squares) or Tg− mice (circles). Data are representative of 3 donors in a total of 22 mice. (C) Accumulation of NK cells developing in B27 Tg+ (squares) and Tg− (circles). Data are representative of 3 donors in a total of 22 mice, from which serial blood samples were obtained (*, p
Immunity. Author manuscript; available in PMC 2017 August 16. Published in final edited form as: Immunity. 2016 August 16; 45(2): 280–291. doi:10.1016/j.immuni.2016.07.005.
Cell-extrinsic MHC class I molecule engagement augments human NK cell education programmed by cell-intrinsic MHC class I Jeanette E. Boudreau1, Xiao-Rong Liu1, Zeguo Zhao1, Aaron Zhang1, Leonard D. Shultz2, Dale L. Greiner3, Bo Dupont1,4, and Katharine C. Hsu1,4,5,*
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1Immunology
Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA 2The
Jackson Laboratory, Bar Harbor, ME 04609
3Program
in Molecular Medicine Diabetes Center of Excellence, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655 4Department 5Weill
of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
Cornell Medical College, New York, NY, 10065, USA
Summary Author Manuscript
The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by “self” MHC class I molecules in a process called “education”. In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B* 27:05+ transgenic mice and patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34+ stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1+ NK cells gained reactivity after adoptive transfer to HLA-B*27:05+ mice or bone marrow chimeric mice where HLA-B*27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell–intrinsic HLA in maintaining NK cell education.
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eTOC Blurb Killer immunoglobulin-like receptors (KIR) and human leukocyte antigens (HLA) calibrate NK cell education. Hsu and colleagues demonstrate that NK cell reactivity increased or decreased,
*
Corresponding author: Dr. Katharine C. Hsu, Department of Medicine, Memorial Sloan Kettering Cancer Center, [email protected]. Author contributions L.D.S. and D.L.G. established the B27 Tg+ mice; J.E.B., X-R.L., A.Z., Z.Z., B.D. and K.C.H. designed and performed experimental analysis; J.E.B., B.D. and K.C.H. wrote and edited the manuscript.
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respectively, when HLA was acquired from neighboring cells or was silenced in educated cells. Therefore, both cell-intrinsic and –extrinsic HLA co-operate to maintain and adjust NK cell education.
Author Manuscript Introduction
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Natural killer (NK) cells integrate activating and inhibitory signals to measure the health of neighboring cells. The major histocompatibility complex (MHC) class I antigens, termed human leukocyte antigens (HLA) in humans are recognized by NK cells as markers of “healthy self” via inhibitory Ly49 receptors in mice or killer immunoglobulin-like receptors (KIR) humans, respectively. Downregulation or loss of class I expression resulting from infection, damage, or transformation, interrupts inhibition and triggers NK cell reactivity through simultaneous activating signals (Long et al., 2013). The capacity of NK cells to recognize cells lacking MHC class I is well established, but the processes by which NK cells are calibrated to recognize “self” MHC class I molecules are not.
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Individual NK cells exhibit variable expression of inhibitory receptors; consequently, NK cells exhibit different sensitivity to inhibition based on the receptor(s) they express and the co-inherited MHC class I (Raulet et al., 2001). A process called “education” calibrates NK cells to recognize and respond to cells that have lost expression of MHC class I (Anfossi et al., 2006; Joncker et al., 2009; Kim et al., 2005). NK cells that express KIR that are specific for “self” MHC class I exhibit greater responsiveness against MHC class I− targets than NK cells that lack inhibitory receptors for “self” MHC class I. Paradoxically, the same interactions between self-specific KIR and MHC class I that program education also govern NK inhibition when the putative target cell expresses the self-MHC class I ligand. Altogether, the reactivity of NK cells is balanced to permit sensitivity to damaged cells without autoimmune consequences. The processes governing NK cell education are not fully understood. In the “licensing” model, NK cells are endowed with functional potential when their inhibitory receptors can Immunity. Author manuscript; available in PMC 2017 August 16.
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engage “self” MHC class I (Anfossi et al., 2006; Kim et al., 2005). In another model, NK cells that lack inhibitory receptors sensitive to “self” MHC class I may be “disarmed” via a process analogous to activation-induced anergy. The capacity for inhibition by “self” MHC class I protects NK cells from anergy, enabling educated NK cells to retain functional capacity (Raulet, 2006).
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The genes for KIR and Ly49 segregate independently from those of MHC class I, and the expression of inhibitory receptors varies among NK cells in an individual’s repertoire (Parham, 2005). As a result, the education of each NK cell is calibrated by its receptor expression and the availability of cognate MHC class I molecules. In mice, NK cell-derived Ly49 molecules interact with cognate MHC class I molecules on both neighboring cells (trans) and on the NK cell itself (cis) (Doucey et al., 2004; Ioannidis et al., 2001; Scarpellino et al., 2007). Via a flexible stalk, Ly49 bends to make the same binding site available to MHC class I ligands in trans and cis (Back et al., 2009; Chalifour et al., 2009; Tormo et al., 1999). Mutation of the stalk to allow trans but not cis binding diminishes NK cell responses to MHC− targets, leading investigators to conclude that Ly49 interactions in cis with MHC class I molecules contribute to mouse NK cell reactivity (Chalifour et al., 2009). Supporting an additional role for trans ligands in mouse NK cell education, mature NK cells adoptively transferred from beta-2 microglobulin (β2m)-deficient mice to MHC class I-sufficient WT hosts results in an increase in NK cell responsiveness (Elliott et al., 2010; Joncker et al., 2010). Reciprocally, NK cells transferred from WT mice to β2m-deficient hosts exhibit decreased reactivity (Joncker et al., 2010). Mixed chimeras of MHC class I moleculesufficient and -deficient stem cells reveal that MHC class I molecules on either hematopoietic or stromal cells can educate mouse NK cells expressing cognate receptor, and responsiveness is highest when MHC class I molecules are present in both compartments (Xie et al., 2010). Together, these findings illustrate that MHC class I molecules expressed by both NK and neighboring cells contribute to NK cell education in mice.
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Although the Ly49 and KIR proteins both educate NK cells, they are the products of distinct non-orthologous gene families (Parham, 2005). Consequently, it has been difficult to translate mouse findings to understand human NK cell biology. Taking cues from the known patterns of NK cell education in mice, we investigated the roles of cell-intrinsic and – extrinsic MHC class I molecules for educating human NK cells. Using a mouse transgenic for HLA-B*27:05, which exhibits the Bw4-ligand for KIR3DL1, we investigated the relative contributions to NK education by HLA class I molecules presented in trans from hematopoietic or stromal cells and present in the NK cell itself. While human NK cell reactivity was augmented by HLA class I available in trans, cell-intrinsic HLA class I was necessary to maintain education. Using RNA interference, we showed that HLA class I molecules on the NK cell itself were required to maintain human NK cell education. HLA class I molecules were readily adopted from trans sources by NK cells after cell transfer, a potential mechanism by which environmental HLA class I molecules can adjust human NK cell reactivity. In patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), we found that donor-derived NK cells analogously exhibit recipient HLA class I molecules on the cell surface, and education that reflects both donor and recipient HLA class I. Altogether, these findings illuminate important aspects in the biology and calibration of
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human NK cell function that may be informative in HCT and adoptive cell therapies, where HLA class I alleles in the donor and recipient may differ.
Results HLA-B*2705 Tg+ and Tg− mice support human NK cell development
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Immunocompromised and humanized mice are increasingly used to study human NK cell function in vivo (Chen et al., 2009; Huntington et al., 2009; Pek et al., 2011). However, these models have not been engineered to express human HLA class I molecules, limiting their utility in investigations of human NK cell development and education. We used a NOD.CgRag1−/−IL2rgtm1Wjl /Sz strain with transgenic expression of human B2M and the HLABw4+ HLA-B*27:05 (B27 Tg+) to understand how interactions with HLA class I molecules impact the education of human NK cells (Figure 1A) (Taurog et al., 1990). Both B27 Tg+ and their Tg− littermates (Tg−) supported the in vivo development of human lymphocytes from CD34+ hematopoietic stem cells, achieving full human chimerism in blood lymphocytes by 7 weeks post-transplant (Figure 1B). Semi-weekly injections of BA/F3 feeder cells that trans-present IL-15 (Pittari et al., 2013) favored human NK cell development in both strains. A greater number of NK cells were recovered from the spleens of B27 Tg+ mice at week nine (Figure 1C) and until at least week 11 (data not shown), suggesting that the presence of HLA class I molecules in trans supports enhanced survival and/or proliferation of NK cells. Humanized mice additionally exhibited monocytes, B and T cells of human origin (Supplemental Table 1).
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NK cells developing from CD34+ stem cells in the humanized mice expressed phenotypic markers similarly to NK cells from the peripheral blood of healthy human donors, with the exception of NKG2A, which was expressed on a majority of NK cells in the humanized mice (Figure 1D–E). CD16, NKG2D and NKp46 were all detectable on the surface of NK cells harvested from the blood and spleen and did not differ between B27 Tg+ and Tg− mice (Figure 1F–I). Inhibitory KIR were expressed on a greater percentage of NK cells developing in Tg− compared with B27 Tg+ mice (Figure 1J–L), consistent with previous studies that demonstrate overexpression of inhibitory receptors in the absence of their ligands (Van Bergen et al., 2013). Similarly, CD57 was expressed at higher frequency among NK cells in Tg− mice (Figure 1M). Human NK cells are educated by cognate HLA-Bw4 ligand in trans
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NK cell effector function is primed in vivo by inflammation, prompted by pathogenassociated molecular patterns (PAMPs) and/or cytokines (Boudreau et al., 2011; Lucas et al., 2007; Strowig et al., 2010). Similar to other humanized mouse models, priming with LPS was necessary for an NK cell response to stimulation with IL-12+IL-18 or the MHC class I− target cell lines, 721.221 or K562; NK cells from unprimed, donor-matched mice were not responsive (Ito et al., 2002; Tanaka et al., 2012) (Supplemental Figure 1). To study NK cell education, we analyzed CD34+ stem cell donors exhibiting cell-surface KIR3DL1, the cognate receptor for HLA-Bw4 (Gumperz et al., 1995). NK cells developed in the humanized mice, including KIR3DL1+ NK cells, degranulated robustly in response to
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K562 cell line targets similar to healthy human donors (Figure 2A). When pairs of Tg− and B27 Tg+ mice were transplanted with CD34+ cells from the same donors, NK cells developing in B27 Tg+ compared with Tg− mice exhibited higher degranulation (Figure 2B). NK cells exclusively expressing KIR3DL1, and therefore lacking KIR2D receptors, exhibited elevated responsiveness (Figure 2C), suggesting that availability of the HLA-Bw4 ligand in B27 Tg+ mice can promote education of developing NK cells expressing the cognate receptor. IFN-γ production was similarly elevated among KIR3DL1+ NK cells from B27 Tg+ mice compared with Tg− animals (Supplemental Figure 2A–B). Donor and recipient HLA class I molecules are required for maximum reactivity
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Reflecting variability in NK cell responses observed between individual humans (Yawata et al., 2006), substantial inter-donor variation in KIR3DL1+ NK cell responsiveness of humanized mice was evident (Figure 2C). If HLA class I molecules contributed by the mouse stromal environment solely determined NK cell education, such variability would not be predicted to occur among our inbred mice. We therefore considered the possibility that the HLA class I molecules from the human stem cell donor might also contribute to NK cell education. To distinguish the educating impact of HLA class I molecules presented by neighboring human hematopoietic cells from HLA class I molecules presented by mouse stromal cells, we examined the cytotoxic and cytokine responsiveness of KIR3DL1+ NK cells that developed in B27 Tg+ or Tg− mice, stratified by the presence or absence of Bw4 in the human CD34+ stem cell donor (Figure 2D, Supplemental Figure 2C). The most responsive cells were KIR3DL1+ NK cells derived from Bw4+ donors and developed in B27 Tg+ mice. KIR3DL1+ NK cells derived from Bw4+ donors and developed in Tg− mice and NK cells derived from Bw4− donors and developed in B27 Tg+ mice exhibited similar responsiveness. The least responsive cells were KIR3DL1+ NK cells derived from Bw4− donors and developed in Tg− hosts. The stepwise escalation of NK cell responsiveness indicates that both donor and host HLA class I molecules influenced NK cell education.
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A contribution of hematopoietic HLA class I molecules to NK cell education was further supported by examination of the KIR3DL1− cells, which demonstrated responsiveness in both Tg− and B27 Tg+ hosts (Figure 2A and D). If HLA class I molecule presentation by mouse stromal cells was solely responsible for NK cell education, we would expect that NK cells expressing KIRs other than KIR3DL1 would be uneducated and hyporesponsive. Therefore, we compared the responsiveness of NK cells expressing KIR2DL2 or KIR2DL3, but lacking other inhibitory KIR, based on the presence or absence of cognate HLA-C group 1 (HLA-C1) ligand in the CD34+ stem cell donor. KIR2DL2+ or KIR2DL3+ NK cells from HLA-C1+ donors developed in either B27 Tg+ or Tg− mice exhibited greater responsiveness compared to KIR2DL2+ or KIR2DL3+ NK cells from HLA-C1− donors (Figure 2E). We conclude that HLA class I molecules presented by donor hematopoietic cells impacts NK cell education. Donor and recipient HLA class I molecules contribute to NK cell education in patients following hematopoietic cell transplantation To understand if our findings in humanized mice reflect human biology, we examined NK cell education in recipients of HLA mismatched hematopoietic cell transplantation (HCT).
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We compared NK cells exhibiting KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 as their sole receptor, stratifying based on the presence or absence of their ligands in the HCT donor and/or transplant recipient. Analogous to the findings in the mouse model, HLA-Bw4 contributed by both the stem cell allograft and the recipient stromal compartment promoted KIR3DL1+ NK cell responsiveness to K562 cells: the highest responsiveness occurred when HLA-Bw4 was available in both (Figure 3A). The educating ligands for KIR2DL1 (HLAC2) and KIR2DL2 or KIR2DL3 (HLA-C1), contributed similarly to NK cell education: ligand present in both the allograft and recipient was associated with the highest reactivity, and similar NK cell responsiveness occurred when the KIR ligand was present in either the stem cell allograft or the recipient (Figure 3B and C). HLA-Bw4 in trans re-educates mature NK cells
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To determine whether NK cell education is a process that persists after egress from the bone marrow, we transferred mature peripheral blood NK cells from the same human donors into pairs of B27 Tg+ or Tg− mice. Human NK cells were detected in the peripheral blood, lymphoid and peripheral tissues for at least 2 weeks after transfer (Figure 4A), and exhibited similar proliferation in B27 Tg+ and Tg− mice (Figure 4B). After two weeks in B27 Tg+ mice, KIR3DL1+ cells exhibited enhanced responsiveness compared with the same NK cells pre-transfer or those incubated in Tg− mice (Figure 4C). Notably, degranulation of mature KIR3DL1+ NK cells did not decrease in an environment lacking HLA-Bw4, indicating that NK cell education was maintained even in the absence of environmental ligand. Gain of function was most readily observed among cells from Bw4− donors transferred to the B27 Tg+ mouse, demonstrating that uneducated NK cells can modulate their education despite being fully mature (Figure 4D). Similarly, KIR3DL1+ NK cells adoptively transferred to B27 Tg+ mice exhibited enhanced IFN-γ production compared with NK cells from the same donor, but transferred to Tg− animals (Supplemental Figure 3). To ascertain if the increased reactivity of NK cells by environmental HLA-Bw4 was specific to KIR3DL1+ cells rather than a general consequence of an altered MHC class I environment, we assessed the reactivity of NK cells solely expressing KIR2DL1, KIR2DL2 or KIR2DL3, comparing the difference in reactivity after transfer from the same donor to B27 Tg+ or Tg− mice (Figure 4E). While KIR3DL1+ NK cells exhibited a 2.5-fold higher increase in effector function in B27 Tg+ mice compared with Tg− mice, KIR2DL1+ and KIR2DL2+ or KIR2DL3+ NK cells had no change in response from pre-transfer (data not shown), and there was no difference in response of KIR2DL1+, KIR2DL2+ or KIR2DL3+ NK cells between mouse strains. Therefore, the upward tuning of NK cell reactivity required cognate HLA class I molecules (Figure 4E and Supplemental Figure 3).
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Gain of function in NK cells was mediated by stromal and hematopoietic cells To better understand the cellular interactions that facilitate the increased function of NK cells by local ligands, we compartmentalized HLA-Bw4 to hematopoietic or stromal cells by creating bone marrow chimeric mice (Figure 5A). Lethally irradiated B27 Tg+ or Tg− mice were reconstituted with whole bone marrow derived from either strain to localize HLA-Bw4 to the donor (D) or recipient (R) compartments (D+R− or D−R+). Tg− mice reconstituted with Tg− bone marrow (D−R−) and B27 Tg+ mice reconstituted with B27 Tg+ bone marrow
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(D+R+) served as controls. After nine weeks, mice had a fully reconstituted hematopoietic compartment. NK cells from healthy human donors were then transferred into these mice to determine whether the hematopoietic (donor), stromal (recipient), or both compartments were responsible for increasing NK cell function. The responsiveness of NK cells derived from the same human donors was thereafter compared in quartets comprised of each combination of HLA-Bw4 localization. Whether HLA-Bw4 was confined to the hematopoietic or stromal cells or present in both, KIR3DL1+ NK cells demonstrated enhanced responsiveness compared to NK cells from the original donor and to NK cells transferred to mice lacking HLA-Bw4 (Figure 5B–C). The responsiveness of KIR2DL2+ or KIR2DL3+ cells from the same donor was not significantly altered in different chimeric constructions (data not shown). We conclude that any trans source of cognate ligands for KIR, hematopoietic or stromal, was sufficient to increase the reactivity of mature NK cells.
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HLA class I molecules are required on NK cells for education
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The observation that NK cell responsiveness persists in the absence of environmental HLA class I after adoptive transfer suggests that maintenance of NK cell education does not require persistent interaction with ligand in trans. We hypothesized that cognate MHC class I molecules on the NK cell are necessary for their function and tested this by introducing shRNA via lentiviral infection to silence expression of β2m in primary NK cells (Figure 6A). Gating for KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 monopositive cells enabled comparison of NK cell function in the presence or absence of normal HLA class I molecule expression. Among each of the KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 monopositive populations, knockdown of β2m induced a significant reduction of responsiveness against K562 cells (Figure 6B). This loss of function was especially profound among educated NK cells. In a representative donor expressing HLA-C1 and HLA-Bw4, β2m silencing diminished the reactivity of educated NK cells expressing KIR2DL2, KIR2DL3 or KIR3DL1 (Figure 6C). After β2m silencing, the reactivity of these NK cells was similar to that of the uneducated KIR2DL1+ population, which was unaffected by loss of β2m. Among 34 different donors, β2m silencing significantly impacted the responsiveness of the educated NK cell populations, where loss of “self” HLA class I molecules led to a 50–60% loss in reactivity (Figure 6D). Mature NK cells can acquire HLA class I molecules from trans sources
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Our findings indicate that HLA class I molecule ligands present in cis maintain NK cell reactivity and in trans augments NK cell reactivity. We hypothesized that trans ligands could be acquired by NK cells. We labeled NK cells from HLA-Bw4− donors with CellTrace Violet (CTV) and measured HLA-Bw4 staining on their membranes after their transfer to B27 Tg+ mice. Within two days of transfer, mouse CD45 was evident on the majority of CTV+ human cells harvested from the blood and organs of both B27 Tg+ and Tg− mice (Figure 7A–B). CTV+ NK cells additionally displayed HLA-Bw4 after incubation in B27 Tg+ but not Tg− mice, demonstrating that NK cells could acquire HLA class I molecules from the recipient (Figure 7C). A similar rate of mouse CD45 uptake was observed among CTV+ cells transferred to both mouse strains (Figure 7D), and HLA-Bw4 uptake was not restricted to KIR3DL1+ NK cells (data not shown). We therefore conclude that cell surface
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protein transfer occurred as a likely consequence of cell-cell interaction, rather than a process mediated by specific receptor-ligand engagement. To determine if HLA-Bw4 acquired from trans sources were associated with gain of function among NK cells, we compared the reactivity of KIR3DL1+ NK cells with or without uptake of HLA-Bw4. CTV+ NK cells isolated from HLA-Bw4− donors staining positive for HLA-Bw4 following transfer to B27 Tg+ mice exhibited enhanced responsiveness compared with HLA-Bw4− NK cells from the same mice (Figure 7E). In contrast, CTV+ NK cells from the same donors transferred to Tg− mice did not exhibit HLABw4 staining or enhanced responsiveness (Figure 7D–E).
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To confirm that these findings were not an artifact of our xenograft model, we examined lymphocytes collected from recipients of HLA-mismatched HCT to discern whether recipient stromal HLA class I molecule might similarly be transferred to donor-derived NK cells in humans. NK cells from patients who had full donor chimerism at one year post-HCT were evaluated. Similar to the mouse studies, NK cells derived from an HLA-Bw4− stem cell donor exhibited HLA-Bw4 expression a year after developing in an HLA-Bw4+ patient (Figure 7F). Altogether, our data support a role for cell-intrinsic HLA class I molecules to maintain NK cell reactive potential and suggest that the requisite ligands might be acquired via membrane transfer from neighboring cells.
Discussion
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Using a humanized mouse transgenic for HLA-B*27:05, we have demonstrated that human NK cell education is shaped by HLA class I molecules present in and around NK cells, where cell-intrinsic HLA class I molecules maintain NK cell reactivity that can be augmented by cognate HLA class I molecules provided by neighboring cells. These findings extend to patients receiving KIR ligand-disparate HCT, in whom reconstituting donorderived NK cells exhibit education that reflects both the donor and recipient HLA class I molecules. KIR3DL1+ NK cells acquired higher responsiveness upon transfer to or following development in an environment replete with cognate HLA class I molecules. Removal of HLA class I molecules from the cell itself by shRNA could diminish the reactivity of an educated NK cell, underscoring a role for cell-intrinsic HLA class I molecules in the maintenance of NK cell education. We found that short interactions were sufficient to transfer HLA class I molecules between mouse stromal cells and transferred human NK cells, and we could identify uptake of recipient HLA class I molecules on donorderived NK cells in HCT patients. It is therefore plausible that trans HLA class I molecules may be recycled for cell-intrinsic interactions.
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Education of human NK cells can occur via HLA class I molecules available in the stromal, hematopoietic and NK cell compartments. For developing and mature NK cells, a source of trans ligand contributed to NK cell education, with the highest reactivity achieved when cognate ligand was available in both trans and cis. NK cell education and maturation were separable, because lack of trans ligand or education did not prevent NK cell differentiation in the Tg− mouse. Similarly, maturation did not prevent subsequent enhancement of NK cell reactivity after transfer of NK cells to B27 Tg+ mice. It is worth noting that the presence of
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cognate ligand in trans alone was insufficient to drive terminal maturation: priming was required before human NK cells developed in vivo were responsive toward MHC class I− target cells. The finding that human NK cells require priming before they exhibit functional education provides insight into human NK cell biology, where testing for the efficacy of unprimed NK cells has not previously been possible. In contrast to observations in mice, we find that educated mature NK cells do not lose responsiveness upon transfer to an environment devoid of HLA class I molecules (Elliott et al., 2010). This finding likely reflects a fundamental distinction in mouse and human NK cell biology that is pronounced in HLA class I-mismatched HCT in patients, where we have demonstrated that NK cell education retained the imprint of donor and recipient HLA class I molecules even a year after HCT.
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Our data reveal that while NK cells could be educated by trans ligands, cell-intrinsic HLA class I molecules played a critical role in the maintenance of NK cell effector potential, a finding supported by the functional consequences of silencing cell-intrinsic HLA class I molecules. The contributions of cell-intrinsic and -extrinsic HLA class I were not mutually exclusive: membrane proteins, including HLA class I, were acquired by adoptively transferred NK cells, leading to acquisition of cis KIR ligands from trans compartments and reflecting a similar process that occurs in mice (Miner et al., 2015). The transfer of membrane proteins is known to occur non-specifically between NK cells and neighboring cells (Carlin et al., 2001; Chow et al., 2013). Contrary to findings in mice, where Ly49 can facilitate uptake of specific MHC ligands (Sjöström et al., 2001), our findings do not support a receptor-specific acquisition of HLA-B*27:05.
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Analogous to mice, where cis interactions between Ly49 and MHC facilitate NK cell education (Bessoles et al., 2013; Chalifour et al., 2009; Doucey et al., 2004; Held and Mariuzza, 2008), cis interactions between KIR and HLA class I molecules may also program NK cell reactivity. Trans ligands may be relevant to NK cell education in at least two ways: by providing a source of HLA class I molecules which, when acquired by NK cells, can be recycled for cis interactions; and/or by providing an inhibitory signal to rescue NK cells from “disarming” via chronic activation (Raulet, 2006). Whether the availability of ligands in trans is sufficient to re-educate NK cells or the acquisition of HLA class I molecules from neighboring cells merely marks NK cells which have engaged them remains to be determined.
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Ly49 and KIR are different molecules that evolved to fulfill remarkably similar roles: establishment of NK cell reactivity and maintenance of self-tolerance. Both molecules are rapidly evolving, and both trigger conserved signal transduction pathways (Kelley et al., 2005; Long et al., 2013). While Ly49 molecules possess a flexible stalk region, which permits cis and trans interactions with MHC via the same binding site, KIR molecules lack such a region, which has made a cis interaction between KIR and HLA class I molecules seem unlikely (Held and Mariuzza, 2008). HLA class I molecules are known to interact in cis with the LILRB1 receptors, however, establishing precedent for a cis interaction in human NK cells that does not rely on a flexible stalk (Li et al., 2013). It is feasible that cellintrinsic HLA class I molecules may bind KIR molecules in cis, to establish a mechanism of
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NK cell education analogous to Ly49 and MHC (Chalifour et al., 2009; Held and Mariuzza, 2008). Environmental changes, including alterations in HLA class I ligand availability for inhibitory receptors, might influence NK cell education. Adjustment would allow real-time re-calibration of effector function for sensitivity to the local environment (Pradeu et al., 2013). Such a mechanism may be beneficial in pregnancy, where upward re-setting of maternal decidual NK cell education by fetal antigens may facilitate trophoblast invasion (Sharkey et al., 2015). NK cell education may also impact outcomes in HCT or adoptive cell therapies, where HLA class I molecules may differ between juxtaposed donor and recipient cells.
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In HCT, gain of NK cell function is only beneficial to the patient. Reports of lower leukemia relapse rates following HLA class I-mismatched HCT may be due in part to enhanced NK cell anti-leukemic surveillance (Ruggeri et al., 2002). Because the risk of relapsed disease following HCT remains disappointingly high, with nearly always fatal consequences, our findings of NK cell education by recipient HLA class I without loss of NK cell education by donor HLA class I support the judicious use of HLA class I molecule-mismatched alternative donor sources. Consideration of HLA mismatched donors for HCT, regardless of haploidentical, umbilical, or unrelated origin, should include donor KIR genotyping to maximize NK cell reactivity.
Experimental procedures Mice
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The Tg(B2M, HLA-B*27:05)56-3Trg allele was generated by injection into the male pronucleus of (C57BL/6J x SJL) fertilized eggs (Taurog et al., 1990). This strain was imported into The Jackson Laboratory and backcrossed onto NOD/Lt until N9 and was then mated to maintain the transgene in the hemizygous state, as it is lethal in the homozygous state. NOD.Cg-Tg(B2M,HLA-B*27:05)56-37Tg/Dvs mice were mated with NOD.CgRag1tm1MomIL2rgtm1Wjl/Sz mice to create the NOD.Cg-Rag1tm1MomIL2rgtmWjl Tg(B2M, HLA-B* 27-05)56-3 mouse strain (B27 Tg+). Expression of B27 on mouse tissues was confirmed by FACS. NOD.Cg-Rag1tm1MomIL2rgtm1Wjl/Sz (NRG) littermate mice (Tg−) were used as controls in all experiments. Mice were maintained in specific pathogen-free conditions and bred in the MSKCC Research animal Resource Center, with amoxicillin and Baytril in food and water given ad libitum. Animal experimentation was conducted in specific pathogen-free conditions and approved by the MSKCC Institutional Animal Care and Use Committee (IACUC).
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Mouse blood and spleen PBMC collection Blood was collected in heparinized tubes by retro-orbital bleeding under isoflurane anesthesia. For timecourse experiments, matched trios of mice with the same transgenic background were engrafted with NK cells from the same donor and alternately sampled to allow a minimum 10-day rest period between bleedings.
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Mice were euthanized by cervical dislocation under isoflurane anesthesia. Spleens were collected in RPMI, dissociated by gentle mashing with a syringe plunger and filtered through 40 μM mesh (BD biosciences, San Jose, CA). Red blood cells were lysed using ammonium-chloride-potassium lysis buffer (ACK) (MSKCC core facilities). Purification and adoptive transfer of human NK cells Umbilical cord blood and buffy coats from healthy human donors were obtained from the New York Blood Center (nybloodcenter.org). Samples were anonymized prior to laboratory analysis; therefore, additional research consent requirements were waived by the MSKCC IRB. Additional PBMC collected from buffy coats, were cryopreserved prior to analysis and stimulated with 1000 IU IL-2/mL overnight prior to activation and FACS analysis.
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DNA was isolated from whole blood using the Qiagen Blood DNeasy kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). KIR typing was performed as described (Hsu et al., 2002; Vilches et al., 2007). A KIR ligand typing kit (Olerup, West Chester, PA) was used to identify individuals exhibiting HLA-Bw4, HLA-C group 1 (HLA-C1) or HLAC group 2 (HLA-C2). NK cells or CD34+ stem cells were isolated by Rosettesep negative selection (Stemcell Technologies, Vancouver, Canada) and confirmed to be >95% pure by FACS. NK cells were adoptively transferred to mice or cryopreserved prior to in vitro analysis. BA/F3 with human IL-15Rα and IL-15
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The mouse pre-B lymphocyte cell line BA/F3 was stably co-transfected with IL-3, IL-15 and IL-15Rα and selected by limiting dilution to create BA/F3 cells that transpresent IL-15 on the IL-15Rα stalk (Pittari et al., 2013). BA/F3-IL15 + IL15Rα stable transfectants were irradiated (100 cGy, cesium-137 irradiator) and 5 × 106 cells were injected intravenously twice weekly to support NK cells in vivo beginning three weeks after CD34+ transplant or immediately after adoptive transfer of NK cells. To prime NK cells derived in vivo, mice were injected with LPS (10 mg/mouse) unless otherwise indicated. Analysis of NK cell proliferation NK cell proliferation was quantitated by CellTrace violet (CTV) dilution (Life Technologies, Eugene, OR). Prior to injection, purified NK cells were labeled with 5 μM CTV. CTV dilution was quantitated in CD56+ cells by flow cytometry (LSR Fortessa, BD Biosciences) and analyzed with FlowJo 9.7.6 software (Ashland, OR). HCT patients and sampling
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All studies using samples from HCT patients were approved by the MSKCC IRB, and all patients consented to the research study. Peripheral blood lymphocytes from 12 patients receiving double-unit cord blood transplantation for the treatment of hematologic malignancies were collected 200–365 days after transplantation, a time at which a dominant donor cord blood unit has engrafted and NK cell reactivity is stabilized (Purtill et al., 2014; Yu et al., 2009). All studied patients were confirmed to have 100% donor chimerism provided by one cord source. KIR genotyping was performed on genomic DNA from cord blood, as described (Hsu et al., 2002). MHC class I allele typing was facilitated by the Immunity. Author manuscript; available in PMC 2017 August 16.
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National Marrow Donor Program, and alleles were assigned to KIR epitope groups using the Immunopolymorphism database (http://www.ebi.ac.uk/ipd/). Cryopreserved PBMC were thawed overnight in RPMI containing 10% FBS and 1000 IU/mL IL-2. PBMC were stimulated with K562 target cells at a 3:1 ratio for 5h and assessed for degranulation within NK cell populations expressing KIR2DL1, KIR2DL2, KIR2DL3 or KIR3DL1 as their sole inhibitory receptor. Stimulation and flow cytometry analysis
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The HLA class I− human target line for NK cells, K562, was grown from mycoplasmanegative stocks and maintained in RPMI containing 10% FBS. NK cells harvested from mice were stimulated using K562 cells (3:1 ratio of PBMC:K562) for 5h in the presence of anti-CD107a (H4A3, BD Biosciences). For measurement of IFN-γ production, 20 ng/mL BFA and 0.03% monensin were added during the final 3.5h of culture to inhibit protein export by NK cells. Non-specific antibody binding was blocked by addition of 1μg/mL total mouse IgG (Sigma-Aldrich, St. Louis, MO). Viable human NK cells were identified by excluding mouse CD45+ cells and dead cells, and gating for human CD56+ cells (live/dead fixable dye, ThermoFisher Scientific, Grand Island, NY). Representative staining and gating are shown in Supplemental Figure 4. All antibodies, clones and sources used to identify NK cell subsets are shown in Supplemental Table 2. Silencing of HLA class I expression
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HLA class I expression was targeted using shRNA against β2m, HLA-B or HLA-C (SigmaAldrich). Cocktails of shRNA were cotransfected with gag-pol and VSV glycoprotein into Phoenix A packaging cells in the presence of 10 μg/mL chloroquine (Naldini, 1998). Control viruses included CFP in lieu of shRNA. Viral supernatants were collected 48–72h posttransfection and transferred to primary NK cells in the presence of 5 μg/mL polybrene and spinfected at 300 × g for 90 min. NK cells were cultured for 48h in RPMI + 10% FBS and 300 IU/mL IL-2 prior to stimulation with K562 cells for functional assessment. Statistical analysis
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Paired samples analysis—To compensate for inter-donor variation and allow for comparison between experiments, paired samples representing the same donor after exposure in vivo to B27 Tg+ or Tg− environments were compared. The fold change in B27 Tg+ or Tg− (%CD107a+ KIR3DL1+ NK cells in B27 Tg+ mice / %CD107a+KIR3DL1+ NK cells in Tg−), or the fold change in in vivo compared with donor (%CD107a+KIR3DL1+ NK cells in B27 Tg+ mice / %CD107a+KIR3D1+ NK cells in donor) was calculated and is displayed in graphs, as indicated. Statistical analyses were completed using paired samples t tests.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments We thank Dr. Thomas A. Waldmann (National Cancer Institute) for cDNA clones for human IL-15 and human IL-15Rα. We thank Dr. Stuart Tangye (Garvan Institute, Darlinghurst, Australia) for providing the mouse pre-B cell line BA/F3, immortalized with mouse IL-3 gene. The authors thank Drs. Joseph Sun and Alan Hanash (Memorial Sloan Kettering Cancer Center) for helpful comments on the manuscript. We are grateful to Chi-Hang Cheung, Jean-Benoît Le Luduec, Shajoti Rahman, and Mimi Tang for technical assistance. This work was supported by NIH P01 (CA23766) and NIH R01 HL088134 to KCH; NIH NCI CA08747 and CA023766 to BD; core grant (CA034196) and NIH R24OOD018259 and UC4DK104218 to LDS and DLG and Core Grant (P30 CA008748) to MSKCC. Additional support was provided from the William H Goodwin and Alice T Goodwin and the Commonwealth Foundation for Cancer Research and the Helmsley Charitable Trust 2012PG-T1D018 (BD).
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Highlights 1.
Human NK cell education reflects cell-intrinsic and -extrinsic HLA class I
2.
NK cells can be educated by HLA class I from hematopoietic and stromal cells
3.
NK cell-intrinsic HLA class I maintains education via interaction with KIR
4.
NK cell function is enhanced after trans-acquisition of cognate HLA class I
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Figure 1. Development and characterization of an HLA class I-expressing humanized mouse model
(A) HLA-Bw4 expression on murine splenocytes. Dashed grey histogram represents B27 Tg+ mice, and the solid black histogram represents Tg− mice. (B) Engraftment of human cells from CD34+ stem cells in B27 Tg+ (squares) or Tg− mice (circles). Data are representative of 3 donors in a total of 22 mice. (C) Accumulation of NK cells developing in B27 Tg+ (squares) and Tg− (circles). Data are representative of 3 donors in a total of 22 mice, from which serial blood samples were obtained (*, p
