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Xenogeneic ₂-Microglobulin Substitution Alters NK Cell Function¹

Loralyn A. Benoît^* and Rusung Tan^2,

^* Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and Department of Pathology and Laboratory Medicine, British Columbia’s Children’s Hospital and University of British Columbia, Vancouver, British Columbia, Canada

	Abstract

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Recently, it has been shown that human ₂-microglobulin (h-₂m) blocks the association between the NK cell inhibitory receptor Ly49C and H-2K^b. Given this finding, we therefore sought to assess the immunobiology of NK cells derived from C57BL/6 (H-2^b) mice expressing exclusively h-₂m. Initial analysis revealed that the Ly49C expression profile of NK cells from h-₂m⁺ mice was modified, despite the fact that H-2K^b expression was normal in these mice. Moreover, the NK cells were not anergic in that IL-2 treatment of h-₂m⁺ NK cells in vitro enabled efficient lysis of prototypic tumor cell lines as well as of syngeneic h-₂m⁺ lymphoblasts. This loss of self-tolerance appeared to correlate with the activation status of h-₂m⁺ NK cells because quiescent h-₂m⁺ transplant recipients maintained h-₂m⁺ grafts but polyinosine:polycytidylic acid-treated recipients acutely rejected h-₂m⁺ grafts. NK cell reactivity toward h-₂m⁺ targets was attributed to defective Ly49C interactions with h-₂m:H-2K^b molecules. With regard to NK cell regulatory mechanisms, we observed that h-₂m:H-2K^b complexes in the cis-configuration were inefficient at regulating Ly49C and, furthermore, that receptor-mediated uptake of h-₂m:H-2K^b by Ly49C was impaired compared with uptake of mouse ₂m:H-2K^b. Thus, we conclude that transgenic expression of h-₂m alters self-MHC class I in such a way that it modulates the NK cell phenotype and interferes with regulatory mechanisms, which in turn causes in vitro-expanded and polyinosine:polycytidylic acid-activated NK cells to be partially self-reactive similar to what is seen with NK cells derived from MHC class I-deficient mice.

	Introduction

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Natural killer cells are critical mediators of acute transplantation rejection as evidenced by their ability to eliminate allogeneic leukocyte grafts in vivo (1, 2) and to lyse allogeneic and MHC class I (MHC-I)³ mutant lymphoblasts in vitro (3, 4, 5). Much of NK cell reactivity can be explained by the missing self-hypothesis which posits that NK cells eliminate target cells that lack sufficient expression of self-MHC-I molecules (6). The missing self-hypothesis has been substantiated by the identification of NK inhibitory receptors in several species that bind MHC-I molecules (7, 8, 9, 10). In mice, NK inhibitory receptors that engage MHC-I belong to the Ly49 family (11). The ligand-binding repertoire of individual Ly49 molecules is somewhat promiscuous and occurs with differing affinity to distinct subsets of MHC-I molecules (11, 12, 13). To date, the best-characterized interactions have been described for Ly49A and Ly49C and their respective associations with H-2D^d and H-2K^b.

The interaction of MHC-I molecules with Ly49 inhibitory receptors is central to the concept of NK cell tolerance. Induction of tolerance occurs during early NK cell ontogeny and involves the acquisition of Ly49 molecules with specificity for self-MHC-I molecules (14, 15, 16, 17). During this stage, binding of Ly49 inhibitory receptors to cognate MHC-I molecules licenses the immature NK cell to complete the maturation process, undergo clonal proliferation, and acquire functional competence (18, 19, 20, 21). NK cells that are unable to transduce Ly49 signals (through lack of Ly49 expression or lack of host MHC-I molecules) remain largely nonresponsive.

The principal mechanism underlying tolerance is the calibration of NK receptor expression by MHC-I. Receptor calibration is thought to adapt the level of receptor expression on the NK cell so that receptor levels can respond to perturbations in MHC-I expression (22, 23, 24, 25). Calibration is achieved by the MHC-I expression profile of the host environment in trans-association as well as by MHC-I molecules expressed by the NK cell in cis-associations (24, 26). Unlike trans-associations which result in inhibition, cis-interactions with MHC-I do not contribute to inhibitory signaling and instead function to lower the activation threshold needed to exceed NK cell inhibition by reducing the number of available Ly49 molecules (26). The balance of these juxtaposed ligands and their cumulative calibration effect on the NK cell determines the effector potential of mature NK cells.

However, the missing self-hypothesis and calibration model fail to explain all patterns of NK cell reactivity and phenotype. One notable example is the effect of human ₂-microglobulin (h-2m) binding to mouse MHC-I H chains H-2D^d and H-2K^b, which has been shown to block their respective associations with Ly49A and Ly49C (27, 28, 29). This restrictive effect has been attributed to the formation of novel surface chemistry and shape that is assumed when the MHC-I H chain binds h-₂m (30, 31), which in turn negatively impacts on the intermolecular association between Ly49 and MHC-I (27, 28, 29, 30, 31, 32, 33, 34). With respect to Ly49C, binding of h-₂m to H-2K^b does not significantly alter the H-2K^b expression level or antigenicity as ascertained using a panel of mAb reagents (29, 33, 34), yet this association renders otherwise syngeneic B6 target cells sensitive to lysis by Ly49C⁺ NK cells (2). Importantly, the susceptibility of h-₂m⁺ MHC-I⁺ targets to otherwise syngeneic LAK is not predicted by the missing self-hypothesis. Thus, this pattern of NK cell reactivity represents a novel model or recognition, distinct from missing self, which we refer to as altered self-recognition. With this in mind, we therefore wanted to assess the NK cell compartment of h-₂m⁺ B6 mice to determine how NK reactivity and regulation might be affected as a consequence of endogenous h-₂m expression.

	Materials and Methods

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Tissue culture and mice

Mouse cell lines P815 (mastocytoma; H-2^d) and YAC-I (T lymphoma; H-2^a) were obtained from the American Type Culture Collection. C57BL/6J (B6) mice were purchased from The Jackson Laboratory. H-2D^b–/– and H-2K^b–/– B6 mice, derived from E14TG2a 129 Ola/Ola embryonic stem (ES) cells (H-2^b/c) (35), were donated by Dr. F. Lemonnier (Pasteur Institute, Paris, France). h-₂m⁺ B6 mice were provided by Dr. J. Chamberlain (Hospital for Sick Children, Toronto, Ontario, Canada) and were generated by microinjecting (B6/SJL)F₂ (H-2^b/s2) single -ell embryos and then backcrossing the offspring onto the B6 Background for 10 generations (34). h-₂m⁺/murine ₂m (m-₂m^–) and h-₂m⁺/H-2D^b–/– mice were generated by breeding h-₂m⁺ B6 mice with m-₂m^–/– B6 mice (38) and then by selectively breeding resulting mice with H-2D^b–/– mice. Note, m-₂m^–/– B6 mice were derived from D3 129 ES cells (H-2^b/c) (36), and the m-₂m^–/– mice used in these experiments were littermates obtained from our breeding regimen. In all experiments, except for the one described in Table III, LAK were generated from either h-₂m⁺/m-₂m^– (transgenic (Tg)) or h-₂m^–/m-₂m⁺ (wild-type (Wt)) mice (lo). Animals were maintained in the specific pathogen-free vivarium at the Ontario Cancer Institute and used according to institutional guidelines. Age-matched female mice, typically 6–8 wk, were used in most experiments. In general, cells were cultured in complete medium: -MEM (Invitrogen Life Technologies) supplemented with 50 µM 2-ME, 10 mM HEPES, and 10% heat-inactivated FBS (Wisent).

5E6 (Ly49C/I), PK136 (NK1.1), H57-597 (TCR), GK1.5 (CD4), 53-6.7 (CD8), KH9 (H2-D^b) and isotype controls were purchased from BD Biosciences. 2.4G2 and Y-3 hybridomas were obtained from the American Type Culture Collection. Y-3 binds the ₁₂ domain of H-2K^b irrespective of the ₂m isoform (37). Production of Y-3 mAb from hybridoma supernatant involved protein G column isolation (Sigma-Aldrich). FITC labeling of Y-3 was performed using FITC-CELITE (Calbiochem).

Staining and flow cytometry

Approximately 2 x 10⁶/ml cells were stained in 100 µl of PBS/1% FBS containing purified 2.4G2 mAb or 2.4G2 hybridoma supernatant for 30 min on ice. Cells were then washed and resuspended in 300 µl of buffer for immediate acquisition on a FACSCalibur (BD Biosciences). Data were analyzed on CellQuest software (BD Biosciences).

h-₂m

Lyophilized h-₂m was purchased from Sigma-Aldrich (purity >90% confirmed by HPLC), dissolved in PBS at 1 mg/ml, and stored at –80°C.

Generation of target cells

Con A lymphoblasts were generated by incubating 10⁷ splenocytes with 2 µg/ml Con A (ICN Pharmaceuticals). Day 1.5–2 Con A blasts were enriched on Lympholyte-M (Cedarlane Laboratories), washed, and then treated for 2 min in 200 mM methyl -D-mannoside/methyl -D-glucoside (Sigma-Aldrich).

Generation of LAK cells

Splenocytes were depleted of RBC by density gradient centrifugation. Adherent cells were depleted using a nylon wool column (38). Eluted cells were incubated with anti-mouse CD4, CD8, and B220 conjugated Dynabeads (Dynal) for 90 min. Immunomagnetic complexes were removed using a Dynal MPC-E magnet. Efficiency of T and B cell depletion was routinely 95%. Approximately 1–3 x 10⁶ NK cells were plated with 10,000 IU/ml human rIL-2 (Proleukin; Chiron) and cultured for 4–11 days. In certain instances, LAK were sorted using a BD FACSVantage (BD Biosciences) 48 h before use.

Cytotoxicity assays

Target cells were labeled for 90 min with 100 mCi of Na⁵¹CrO₄ (NEN Life Science). LAK were serially diluted and plated as 100-µl aliquots into 96-well plates, to which was added 100 µl of target cells (20,000/ml). Plates were incubated for 4 h, after which supernatant was harvested and counted using a counter. Percent specific lysis was calculated by averaging the gamma emission from five replicate wells and calculated as follows: 100 x (experimental release – spontaneous release)/(maximum release – spontaneous release). Spontaneous release refers to supernatant from targets cultured alone, maximal release refers to supernatant from targets incubated in 1% glacial acetic acid, and experimental release refers to supernatant obtained at each E:T ratio. Lytic unit (LU) analysis was performed according to Bryant et al. (39).

Preparation and infusion of donor cells

Leukocytes were labeled with 1 µM carboxyfluoroscein diacetate, succinimidyl ester (BioCan Scientific) for 10 min at 37°C in serum-free PBS and then washed in complete medium. Labeled cells (3.0 x 10⁷) were injected into the lateral tail vein of recipient mice. In vivo, NK activation was induced by i.p. administration of 100 µg of polyinosine:polycytidylic acid (poly I:C; stock 333 µg/ml in PBS; Sigma-Aldrich). Inguinal and axillary lymph nodes (LN) were harvested at 48 h postinfusion and single-cell suspensions were analyzed by FACS to determine the relative percentage of CFSE⁺ leukocytes.

	Results

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Phenotypic analysis of NK cells from h-₂m⁺ B6 mice

Since it has been shown that binding of h-₂m impedes the recognition of H-2K^b by Ly49C⁺ NK cells, we sought to characterize how Tg expression of h-₂m might influence the NK cell compartment. To begin with, we found that splenic NK cells (NK1.1⁺TCR^–) were more abundant in 5-wk-old h-₂m⁺ mice compared with controls (cf 4.2% vs 2.1%; p < 0.05; Fig. 1A and Table I). Furthermore, h-₂m⁺ splenocytes demonstrated an increased frequency of Ly49C⁺ NK cells (cf 57% vs 38%; p < 0.001; Fig. 1A and Table I) as well as increased Ly49C mean fluorescence intensity (MFI) staining (cf MFI values 446 vs 228; p < 0.0005; Table I). In vitro lymphokine-activated NK cells, or LAK, where also assessed: h-₂m⁺ LAK exhibited similarly elevated numbers of Ly49⁺ NK1.1⁺ cells (cf 46% vs 36%; Fig. 1B) and an increased Ly49C expression level (cf MFI values 226 vs 102; Fig. 1B).

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Representative phenotypes of NK and LAK cells from 5-wk-old h-2m+ mice. A, Wt and h-2m+ splenocytes were stained for NK1.1, TCR-, and Ly49C. B, Wt and h-2m+ LAK were stained for NK1.1 and Ly49C. Analysis was performed using FACS.

View this table: [in this window] [in a new window]   Table I. NK cell phenotype of 5-wk-old h-2m+ B6 micea

View this table: [in this window] [in a new window]   Table II. NK cell phenotype of older h-2m+ B6 micea

Lytic activity of h-₂m⁺ NK cells in vitro

To test for anergy, we assessed the oncolytic capacity of h-₂m⁺ LAK using prototypic tumor targets. Wt and h-₂m⁺ bulk LAK demonstrated equal spontaneous cytotoxic killing potential against YAC-I targets (Fig. 2A). We also assessed activation-enhanced killing using P815 as target cells and the mAb NK1.1, which binds to the NK activation receptor NKR-P1c. As depicted in Fig. 2B, Wt and h-₂m⁺ bulk LAK demonstrated nearly identical killing profiles for both untreated and anti-NK1.1-treated targets. Next, we assessed the killing potential of h-₂m⁺ LAK against syngeneic lymphoblasts. As expected, Wt and h-₂m⁺ LAK were essentially nonreactive toward Wt lymphoblast targets (Fig. 3, A and B). However, we unexpectedly found that both the Wt and h-₂m⁺ effector preparations lysed h-₂m⁺ target cells (Fig. 3, A and B), although lysis mediated by h-₂m⁺ LAK was 2-fold less compared with lysis mediated by Wt LAK (cf LU₃₀ values 57.4 and 25.4).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Oncolysis of prototypic tumor cells by h-2m+LAK. Wt and h-2m+ LAK were used as effectors against YAC-I (A) and P815 (B) in the presence or absence of anti-NK1.1.Error refers to SEM.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Cytolysis of syngeneic lymphoblasts by h-2m+ LAK in vitro. Bulk LAK generated from Wt (A) and h-2m+ (B) mice were evaluated for their ability to lyse Wt and h-2m+ Con A blasts. C, H-2Db–/– and H-2Db–/–h-2m+ LAK were sorted into Ly49C–NK1.1+ and Ly49C+NK1.1+ populations and used as effectors against H-2Db–/– and H-2Db–/–h-2m+ Con A blasts. Error refers to SEM.

An important consideration is the fact that the mice used in theses experiments were initially derived using ES cells of 129 (H-2^b/c) and B6/SJL (H-2^b/s2) origin before the ensuing litters were backcrossed onto the B6 background (34, 35, 36). To rule out the possibility that genetic contaminates might be contributing to the effects depicted in Fig. 3, Wt and h-₂m⁺ lymphoblast targets that were either untreated or pulsed with 10 µg/ml h-₂m for 2 h (Fig. 4) were tested for relative sensitivity to LAK-mediated killing. We found that lysis of h-₂m⁺ targets was almost identical to the lysis values obtained when target cells were pulsed with exogenous h-₂m, thereby indicating that h-₂m was primarily responsible for the heightened target cell sensitivity.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Confirmation that loss of inhibition is due to h-2m. Wt (A) and h-2m+ (B) LAK were used as effectors against Wt and h-2m+ Con A blasts that were either untreated or pulsed with 10 µg/ml exogenous h-2m+. Error refers to SEM.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Cytolysis of MHC-I mutant target cells by syngeneic LAK in vitro. H-2Kb–/– (A), H-2Db–/– (B), 2m–/– (C), and h-2m+ (D) LAK were assessed for their relative ability to lyse both syngeneic and Wt Con A blasts. Experiments in A–C were repeated twice, D was repeated four times. Error refers to SEM.

Lytic activity of h-₂m⁺NK cells in vivo

Although the results depicted in Figs. 3–5 indicate that under conditions of IL-2 activation, h-₂m⁺ NK cells can lyse normally self-tolerant tissues, h-₂m⁺ mice housed under specific pathogen-free conditions did not exhibit gross signs of autoimmune processes. Therefore, to assess the physiological relevance of our findings, we examined NK cell self-tolerance in vivo. In this regard, Wt, h-₂m⁺, and RAG-2^–/– mice were either untreated (Fig. 6A) or poly(I:C) treated (Fig. 6B), then were infused with CFSE-labeled splenocytes derived from Wt, h-₂m, and ₂m^–/– mice. In the untreated cohort, Wt recipients moderately rejected both h-₂m⁺ and ₂m^–/– grafts, as the percentage of CFSE⁺ cells was 1.6 (p < 0.0001)- and 1.7 (p < 0.0001)-fold lower, respectively, relative to Wt graft values. Untreated h-₂m⁺ recipients failed to reject syngeneic grafts (1.1-fold increase, p < 0.2), although they partially rejected ₂m^–/– grafts (1.7-fold increase, p < 0.0001). However, a distinct pattern of rejection was observed in mice that were treated with poly(I:C), a synthetic analog of dsRNA that activates NK cells through induction of IFN synthesis (41). We found that all treated recipients, including h-₂m⁺ hosts, efficiently rejected both h-₂m⁺ and ₂m^–/– grafts, although rejection of h-₂m⁺ grafts by Wt and RAG-2^–/– recipients was more profound (relative to Wt graft rejection, 110- and 90-fold, respectively) compared with the rejection mediated by h-₂m⁺ hosts (3-fold, p < 0.000001; Fig. 6B).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Rejection of h-2m+ leukocytes in vivo. In brief 3.0 x 0E7 CFSE-labeled splenocytes from Wt, 2m–/–, and h-2m+ (Tg) mice were infused into Wt, h-2m+ (Tg), and RAG-2–/– recipients that were either untreated (A) or poly(I:C) (B) treated. LN were harvested at 48 h and assessed for percentage of CFSE+ cells using FACS. Error refers to SEM.

Having assessed the NK cell compartment of the h-₂m⁺ mouse, we wished to also examine how h-₂m-bound MHC-I molecules could affect NK cell regulation. Note, there are two basic methods by which MHC-I can regulate Ly49 function. The first is in trans-associations whereby MHC-I molecules on the target cell associate with Ly49 molecules on the surface of the NK. The second method is by cis-associations whereby MHC-I molecules expressed on the surface of the NK cell bind to Ly49 molecules on the same cell (26). In relation to cis-regulation, it is important to consider that MHC can be expressed on the NK cell surface as a consequence of cell-autonomous biogenesis or it can be appropriated from the surface of a target cell via passive mechanisms such as MHC-I shedding (42) or actively using specific receptors (43, 44). With this in mind, we therefore sought to determine whether h-₂m-bound H-2K^b molecules could participate in cis-regulation of Ly49C. To do this, we created a genetic titration matrix of effector and target cell combinations using Wt and h-₂m⁺ mice mated to generate F₁ progeny. Parental and F₁ LAK were then used as effectors against syngeneic Con A targets. The results are summarized in LU per 10⁶ effector cell values in Table III with corresponding descriptions of the cis-/trans-associations. For trans-associations, a gene titratable affect was measured whereby all LAK preparations lysed target cells according to the target cell hierarchy: Wt << F₁ < h-₂m⁺. In contrast, for cis-associations, the LAK preparations lysed target cells according to the effector cell hierarchy: h-₂m⁺< F₁ <<Wt. This pattern of reactivity is consistent with the interpretation that h-₂m impairs the ability of Ly49 to bind MHC-I in cis-associations, in addition to its ablative effect in trans-associations.

Previously, it has been shown that Ly49A can selectively take up H-2D^d molecules from the target cell surface and that these acquired molecules can regulate NK cell function through binding to Ly49A in cis (32, 43, 44). We accordingly wanted to assess Ly49C-mediated uptake of H-2K^b and to determine whether it might be influenced by the expression of h-₂m on target cells. To test this, H-2K^b–/– LAK were coincubated with either Wt or h-₂m Con A blasts for 3.5 h and then assessed for de novo H-2K^b expression based on whether the LAK were Ly49C^–NK1.1⁺ or Ly49C⁺NK1.1⁺ (Fig. 7). Significantly, we found that Ly49C⁺H-2K^b–/– LAK that had been coincubated with either Wt or h-₂m targets demonstrated an increase in H-2K^b MFI staining (baseline MFI 9.5, cf with Ly49C^– H-2K^b–/– LAK MFI 27.4), where a preferential increase was seen for Ly49C⁺H-2K^b–/– LAK that had been incubated with Wt target LAK (MFI, 43.7) compared with Ly49C⁺LAK that had been coincubated with h-₂m⁺ targets (MFI, 31.9).

View larger version (29K): [in this window] [in a new window]   FIGURE 7. h-2m impedes NK inhibitory receptor-mediated uptake of MHC-I. Wt and H-2Kb–/– LAK were coincubated with Wt or h-2m+ Con A blasts for 3.5 h. Cells were harvested, treated with 2.4G2, and then stained with anti-Ly49C, NK1.1, and H-2Kb for analysis by FACS. Histogram analysis was performed using NK1.1+Ly49C+ and NK1.1+Ly49C– gated populations.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Differential NK inhibitory receptor-mediated uptake of MHC-I in vivo. H-2Kb–/– hosts were infused with 3.0 E7 LN cells from Wt (A and B) and h-2m+ (C and D) mice. Recipients were either untreated (A and C) or administered poly(I:C) (B and D). After 18 h, animals were sacrificed and splenocytes pretreated with 2.4G2 were assessed for H-2Kb transfer to NK1.1+ cells using FACS. Histograms are representative of n = 3.

	Discussion

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The first bona fide assessment of "altered self" examined the role of peptide in NK cell recognition by Ly49 molecules in vitro (45). In particular, Su et al. (45) demonstrated that replacing low-affinity peptides with exogenous, high-affinity peptide (pOVA) blocked the ability of Ly49C to functionally engage H-2K^b such that B6 target cells were rendered sensitive to syngeneic NK-mediated lysis. The authors concluded that NK cells bind to MHC-I molecules that are bound by lower-affinity self-peptides via select Ly49 molecules. More recently, we have demonstrated a role for ₂m in Ly49C recognition of H-2K^b in vitro and in vivo where inclusion of h-₂m prevented recognition of H-2K^b by syngeneic Ly49C⁺ NK cells without significantly altering MHC-I expression profiles (29, 33, 34). From this we concluded that NK cells can distinguish between self- and modified self-H-2K^b molecules via Ly49C.

In that these two experimental models suggest that NK cells are capable of recognizing or responding to altered self, we were interested in reassessing the altered self-model using the h-₂m Tg B6 mouse to determine whether h-₂m (altered self) could influence NK cell biology. Initial phenotypic analysis of young mice revealed that the percentage of NK1.1⁺ splenic NK cells were subtly increased in the Tg setting, as well as the percentage and MFI staining of Ly49C (Fig. 1 and Table I). The fact that significant levels of Ly49C⁺ NK cells exist in the Tg mice is evidence that mechanisms other than clonal deletion are responsible for tolerance in these mice. Furthermore, since expression of Ly49C in h-₂m⁺ mice appears to be up-regulated, our results are consistent with the calibration model of receptor regulation (22) and suggest that the NK compartment of h-₂m⁺ mice responds to altered self in much the same way as NK cells from MHC-I-deficient mice respond to missing self. In contrast, immunophenotypic analysis of older h-₂m⁺ mice revealed a reduction in Ly49C expression below control values (Table II). We speculate that the reduction of Ly49C expression over time reflects the importance of "inhibitory" signaling in the periphery such that in the absence of signals transduced through Ly49 receptors, mature NK cells either die or remain relatively nonproliferative, a property that seemingly parallels the requirement for Ly49 engagement by MHC-I molecules seen during early NK cell ontology (18, 19, 20).

We next assessed the functionality NK cells derived from h-₂m⁺ Tg mice. We found that LAK derived from h-₂m⁺ mice retained full capacity to lyse tumor targets in response to NK1.1 ligation and missing self-recognition (Fig. 2) and remarkably demonstrated self-reactivity toward syngeneic lymphoblast targets (Fig. 3B). Using fractionated LAK and targets deficient in H-2D^b, this breech in tolerance was restricted to the Ly49C⁺ NK population (Fig. 3C). From these findings, we concluded that h-₂m⁺ LAK are not functionally anergic in that they can be made to breach tolerance in response to IL-2 stimulation. However, in that the breech in self-tolerance was not anticipated based on the bulk of earlier reports concerning missing self-recognition (3, 5, 46, 47, 48, 49), we reassessed this finding using LAK derived from H-2K^b–/–, H-2D^b–/–, and ₂m^–/– mice against syngeneic lymphoblast targets and found that a similar in vitro pattern of reactivity could be observed using other MHC-I mutant systems as well (Fig. 5). We therefore conclude that the effect of h-₂m on NK cell development is similar to the effect of MHC-I deficiency in that altered self-NK reactivity can be partially normalized in vitro using IL-2 (17).

We speculate that this discrepancy with the reported literature is largely attributable to the quality of the effector populations used in the assays. In particular, many of the previous reports have used unpurified poly(I:C)-activated splenocytes, whereas we have used highly purified (95% NK1.1⁺CD3^–) IL-2 activated NK cells. Admittedly however, our observation of partial NK self-tolerance in MHC-I mutant mice using IL-2 is not entirely unprecedented; there are a few reported cases that document variations of this phenomenon as well. The earliest account involved the use of IL-2-activated LAK and Con A lymphoblast targets for in vitro assessment of NK reactivity profiles. Using this system, the authors identified a low level (10% specific lysis at 100:1 E:T) of self-reactivity using B6 mice (50), a finding which has since been consistently reported in the literature. Similarly, BALB.B (H-2^b)-derived LAK have been reported to exhibit significant (30% specific lysis at 100:1 E:T) self-reactivity toward Con A lymphoblasts (51). Indeed, this pattern has been extended to human NK cells as well; Zimmer et al. (52) have shown that LAK generated from TAP-deficient patients are efficient at lysing MHC-I-deficient target cells, although unstimulated NK cells from this cohort appear to be functionally anergic. Therefore, there is historical evidence that suggest that IL-2 can cause NK cells from both Wt and MHC-I mutant cells to partially breech tolerance in vitro.

To confirm the in vivo relevance of our functional data, we applied a model of leukocyte transplantation that would enable dissection of NK cell function under both quiescent and activated states. We found that quiescent h-₂m⁺ hosts fully accepted h-₂m⁺ but that h-₂m⁺ hosts that were activated using poly(I:C) acutely rejected h-₂m⁺ transplants almost to the same degree as they rejected ₂m^–/– leukocyte grafts. A comparable finding of differential MHC-I-deficient graft rejection under quiescent and poly(I:C) administration has been previously reported (21). We therefore concluded that NK cell reactivity toward altered self can be modified, much like missing self-recognition, depending on the activation status of the NK cell.

The combination of our data obtained using in vitro IL-2-activated LAK and the in vivo NK cell model addresses a long-standing oversight in the literature concerning self-tolerance in the context of missing self-recognition. In particular, our results show that MHC-I mutant NK cells exposed to either IL-2 or type I IFN are partially released from anergy such that they can be made to lyse syngeneic targets. In that this effect is only partial relative to the effect measured for Wt LAK using the same target cells, it appears that IL-2 and IFN are not simply reversing anergy or forcing maturation of developmentally static NK cells. Therefore, additional work should be undertaken in the future to explore the effects of IL-2 on modulating NK target cell specificity. Of interest would be the effects of systemic inflammation on NK cell tolerance in MHC-I mutant mice to determine whether this is a physiologically relevant behavior. In this line, there is some evidence for accelerated autoimmunity (lupus) in mice deficient in ₂m (53).

The underlying mechanism that accounts specifically for the effect of h-₂m substitution on Ly49C recognition of H-2K^b can be explained using several different models. One possible explanation is that binding of h-₂m to the murine MHC-I H chain is changing the repertoire of peptides that bind to the MHC molecules and that is in turn is altering the manner in which Ly49C engages H-2K^b. The argument against this model comes from structural data by Dam et al. (32) illustrating Ly49C in contact with H-2K^b/pOVA/m-₂m. Using analytical sedimentation analysis, the authors demonstrated that substitution of m-₂m with h-₂m impeded the association between Ly49C and H-2K^b/pOVA. Similarly, using MHC-I tetramer analysis of the Ly49-binding repertoire, Michaelsson et al. (27) demonstrated that H-2K^b/pOVA tetramers refolded with m-₂m but not h-₂m could bind to Ly49C⁺ cells. In both cases, H-2K^b/pOVA complexes could efficiently bind Ly49C only when they were bound by m-₂m but not with h-₂m. Relating this back to the cocrystal structure, Dam et al. (32) attributed this finding to specific m-₂m amino acid residues that pair with Ly49C (32). In particular, absence of these pairing residues in h-₂m results in loss of critical chemical interactions that in turn are theorized to weaken the association between h-₂m/H-2K^b and Ly49C. Although it remains possible that the ₂m substitution can alter the repertoire of bound peptide (as is theorized by Perarnau et al. (35)), we can safely say from Dam et al. (32) and Michaelsson et al. (27) that this is not a primary contributing factor in our system. In support of our position, we have shown that h-₂m does not alter the ability of H-2K^b to bind to exogenous pOVA (data not shown) and furthermore, that the TCR V repertoire in these h-₂m Tg mice is not significantly altered as a consequence of the xenogeneic substitution (data not shown), suggesting that the effects of h-₂m on the peptide-binding repertoire are likely to be subtle.

Finally, we examined how the expression of h-₂m could influence basic methods of receptor-mediated regulation of NK cell function. Our reasoning for undertaking these experiments was based on the fact that the binding of h-₂m to H-2K^b inhibits recognition by Ly49C in trans, thus we wanted to determine whether cis-associations were similarly affected. To do this, we first compared the ability of LAK derived from Wt (m-₂m only), h-₂m Tg (h-₂m only), and F₁ progeny (combined m-₂m and h-₂m) to kill all three target sources. We found that h-₂m impaired Ly49C-mediated recognition of H-2K^b in both the cis and trans configurations. As well, comparing the nominal effect of the half m-₂m gene dosage in cis where Wt H-2K^b molecules present on the F₁ LAK with the null m-₂m gene dosage effect in cis seen for lysis of Tg targets by Tg LAK (cf LU values 107.5 and 100.0) and the sizable effect of the full m-₂m gene dosage in cis seen in the lysis of Tg targets by Wt LAK (LU value 294.1), we conclude that the trans-associations exert a more profound regulatory effect compared with the cis-associations. This finding is discordant from predictions made from the cocrystal structure of Ly49C complexed with H-2K^b, in that the topology detailed therein is suggestive of cis-association, thereby implying that the dominant configuration for the Ly49C/H-2K^b system is in cis, not in trans (32). We believe that our discordant findings may relate to the choice of high-affinity peptide used in the cocrystal structure and/or may be a consequence of the bacterially expressed recombinant products used in the assessment inasmuch as glycosylation of the ₂ domain has been shown to positively contribute to trans-associations (54) and influence tetramer binding (12). Thus, this crystal structure, we believe, may have reported a bias in its configuration.

Having shown that cis-associations were similarly impaired by h-₂m, we next assessed whether h-₂m could impede receptor-mediated uptake of H-2K^b (in trans) so as to influence NK cell regulation in cis. To this end, we found that h-₂m impeded the selective uptake of H-2K^b molecules by Ly49C, although on account of the relatively high background staining, we could not exclude other mechanisms as well. Importantly, our results can be applied to understanding how NK cells can be made to lyse altered self inasmuch as cis-regulation is impaired (in the context of h-₂m) and cannot be efficiently remedied by exposing NK/LAK to h-₂m⁺ tissues/target cells due to impaired receptor-mediated uptake of H-2K^b.

With respect to the high background staining occurring in the context of selective MHC-I transfer (Fig. 7), we essentially ruled out the possibility that FcRs drove up the background staining of H-2K^b because we performed our surface stains in the presence of the Fc block mAb 2.4G2 (purified or hybridoma supernatant). 2.4G2 binds with high affinity to CD16 and CD32, the key FcRs expressed on the NK1.1⁺ gated fraction. We feel that a more likely explanation for the elevated "background" MHC-I transfer may be due to passive MHC-I shedding (42). As well, ₂m (either mouse or bovine derived) present in vitro or in vivo may be recombining with the Tg H-2K^b molecules and thus facilitating transfer. In particular, bovine ₂m present in the assays may be relevant to our system in that it has previously been shown that bovine ₂m present in culturing serum can contribute to the formation of a viable interface for Ly49C binding to H-2K^b (29). A third possibility is that there is another (unknown) Ly49 molecule that is equally represented in the Ly49C^+/– fractions that can bind with sufficient affinity to H-2K^b bound by h-₂m and can mediate uptake of these hybrid H-2K^b molecules.

We next tested selective MHC-I uptake in vivo under quiescence or activation using H-2K^b–/– recipients transplanted with either Wt or h-₂m⁺ leukocyte grafts. In this scenario, we observed a variable pattern of MHC uptake, depending on the activation status of the host as well as the xenoform of ₂m (Fig. 8). In particular, it appeared that activated NK cells uniformly engage target cells and actively appropriate target cell MHC-I molecules while under quiescent conditions; only a portion of the host NK were thus licensed appropriated target cell MHC-I in a less selective manner. Our results indicate that NK cells exhibited diminished ability to take up hybrid molecules; 20% less in the quiescent hosts and 55% less in the activated hosts, thereby suggesting that the reduced regulation in cis attributed to h-₂m-bound-H-2K^b molecules may account, in part, for the increased NK-mediated rejection of h-₂m⁺ syngeneic grafts depicted in Fig. 5.

One limitation to our experimental system was that we have had to rely on the mAb (SW)5E6 to denote and sort for Ly49C. The issue with mAb 5E6 is that it recognizes both Ly49C and the B6 allele of Ly49I (55). Unfortunately, at this time, the Ly49C-specific mAb 4LO-3311 is no longer available and thus there is no experimental solution to this problem. In that Ly49I can bind 5E6, changes in Ly49I expression and function may in part explain our results. However, we disfavor this possibility in that it has been previously shown, using Ly49I-transfected COS-7 cells, that Ly49I does not effectively engage any MHC elements or B6 (H-2^b) origin (45).

In conclusion, these data detail a model of altered self-recognition whereby xenogeneic substitution of ₂m affects both the ability of the NK cells to recognize MHC-I as well as NK cell biology. In particular, mice that express h-₂m demonstrate a pattern of reactivity that closely parallels the NK-reactivity pattern seen in MHC-I-deficient models of NK tolerance. The fact that NK cells from these Tg mice can be made to breach tolerance in response to cytokines such as IL-2 and IFN- implies a potential role for NK-mediated autoimmunity under inflammatory conditions in MHC-I-deficient or altered MHC-I mice, with possible implications for allo-/xenotransplantation. These data suggest that there is merit in further investigating the terms of autoaggressive NK cell activity, as well as the underlying mechanism of NK cell tolerance in the context of missing or altered self.

	Acknowledgments

 
This work was assisted through the use of research facilities maintained by A. S. Manukian, R. G. Miller, J. R. Woodgett, J. M. Penniger, and M. Colonna.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ R.T. is a Michael Smith Foundation Senior Scientist and is funded by the Canadian Institutes of Health Research.

² Address correspondence and reprint requests to Dr. Rusung Tan, Department of Pathology and Laboratory Medicine, British Columbia Children’s Hospital, 4480 Oak Street, Room 2G5, Vancouver, British Columbia, Canada. E-mail address: roo{at}interchange.ubc.ca

³ Abbreviations used in this paper: MHC-I, MHC class I; ₂m, ₂-microglobulin; m, mouse; h, human; MFI, mean fluorescent intensity; LAK, lymphokine-activated killer; poly(I:C), polyinosine:polycytidylic acid; Wt, wild type; Tg, transgenic; LU, lytic unit; LN, lymph node.

Received for publication December 22, 2006. Accepted for publication May 16, 2007.

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