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Tolerance and Alloreactivity of the Ly49D Subset of Murine NK Cells¹

Thaddeus C. George^*, John R. Ortaldo, Suzanne Lemieux, Vinay Kumar^* and Michael Bennett^2,*

^* Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235; Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702; and Human Health Research Center, Institut National de la Reserche Scientifique-Institute Armand-Frappier, University of Quebec, Laval, Canada

	Abstract

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Class I-specific stimulatory and inhibitory receptors expressed by NK cell subsets contribute to the alloreactive potential of the self-tolerant murine NK cell repertoire. In this report, we have studied potential mechanisms of tolerance to the function of the positive signaling Ly49D receptor in mice that express one of its ligands, H2-D^d. Our results demonstrate that H2-D^d-expressing mice possess a large Ly49D⁺ subset of NK cells that is functionally capable of rejecting bone marrow cell (BMC) allografts in vivo and lysing allogeneic Con A lymphoblasts in vitro. Also, we show that the Ly49D receptor is responsible for the ability of H2^b/d F₁ hybrid mice to reject H2^d/d parental BMC (hybrid resistance). Thus, deletion or anergy of Ly49D⁺ cells in H2-D^d+ hosts cannot explain self tolerance. Our functional studies revealed that coexpression of the D^d-specific Ly49A or Ly49G2 inhibitory receptors by Ly49D⁺ cells resulted in tolerance to D^d+ targets, while coexpression of K^b-specific inhibitory receptors Ly49C/I resulted in tolerance to K^b+ targets. Only in H2^d/d cells did Ly49C/I dominantly inhibit Ly49D-D^d stimulation. This correlated with an increased mean fluorescence intensity of Ly49C expression, as well as an increased percentage of Ly49C⁺ cells in the Ly49D⁺A/G2^- compartment. Therefore, we conclude that self tolerance of the Ly49D subset can be achieved through coexpression of a sufficient level of self-specific inhibitory receptors.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Natural killer cells mediate acute rejection of bone marrow cell (BMC)³ allografts in vivo and lysis of tumor cells and allogeneic lymphoblasts in vitro (1, 2, 3, 4). Many patterns of NK-mediated alloreactivity can be explained by the missing self hypothesis, which contends that tolerance of the host NK cells requires target cell expression of all of the class I molecules that the host considers self (5). Thus, the missing self hypothesis predicts the rejection of parental BMC grafts by H2 heterozygous F₁ hybrids (hybrid resistance), and the rejection of B6 BMC grafts by B6 hosts transgenic for the D^d alloantigen (D8 strain) (6, 7, 8, 9, 10). In mice, members of the class I binding Ly49 family of receptors, expressed on partially overlapping subsets of NK cells, are responsible for the specificity of a particular NK cell within the repertoire (11, 12). Some members of this family, including Ly49A, C, G2, and I, contain a cytoplasmic immunoreceptor tyrosine based inhibitory motif (ITIM) (13). Upon receptor cross-linking, the phosphatase SHP-1 is recruited to the phosphorylated ITIM, resulting in dominant inhibition of NK cell function (14, 15, 16, 17). Ly49A and Ly49G2 transmit inhibitory signals from H2-D^d, while Ly49C and Ly49I transmit inhibitory signals from the K^b Ag.⁴ As a result, D^d-negative target marrow or lymphoblastic cells may be susceptible to rejection or lysis by Ly49A⁺ or G2⁺ NK cells, while K^b-negative targets may be susceptible to Ly49C⁺ or I⁺ NK cells. The class I molecules expressed by the host influence both the percentages and cell surface expression levels of self-class I-specific inhibitory Ly49 receptors, such that all NK cells in the host are self tolerant, yet sensitive to perterbations in self class I (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). It follows that donor BMC susceptible to rejection do not express sufficient class I ligands capable of inhibiting all of the recipient’s NK cell subsets.

Little is known about the murine receptor-ligand interactions responsible for triggering NK cell activation. Evidence from other species demonstrated that class I molecules can serve as stimulatory ligands for NK cells. In the rat system, both positive and negative recognition of class I molecules is readily detectable both in vitro and in vivo (30, 31, 32). Furthermore, members of the human Ig superfamily of class I-specific receptors either stimulate or inhibit NK cell function (33). Inhibitory members, like inhibitory members of the murine Ly49 family, have a cytoplasmic ITIM (34, 35). Stimulatory members, which lack the ITIM, instead coassociate with an immunoreceptor tyrosine-based activation motif containing DAP12 molecule (36). Recently, the murine Ly49D receptor, which lacks a cytoplasmic ITIM, was also shown to associate with DAP12 (37, 38, 39). Cross-linking of the Ly49D/DAP12 complex results in stimulation of an intracellular kinase cascade and activation of NK cell function (38, 39, 40). Three lines of evidence demonstrate that Ly49D stimulates NK function upon interacting with specific target cell class I ligands. In vivo administration of anti-Ly49D mAbs reversed the rejection of D8 BMC by B6 host NK cells (41). In vitro blocking with anti-Ly49D abrogates the lysis of D^d, D^r, and D^sp2 expressing lymphoblasts by sorted and activated Ly49D⁺ B6 NK cells (42). Finally, the rat NK cell line (RNK-16) transfected with Ly49D specifically lyses tumor targets transfected with H2-D^d, unless either the killer cell is preincubated with F(ab')₂ anti-Ly49D or the target cell is incubated with F(ab')₂ anti-H2-D^d (43). D8 targets were found to be susceptible to lysis by the small subset of B6 NK cells that expressed Ly49D but lacked expression of inhibitory Ly49 receptors specific for D^d and K^b class I molecules on the D8 cell (Ly49A, C, G2, I) (42). Thus, the presence of small subsets of NK cells that express stimulatory but not inhibitory receptors specific for target cell class I ligands can be responsible for the rejection of BMC allografts.

The existence of class I triggering receptors can explain at least some instances of NK-mediated allorecognition. Furthermore, their existence also necessitates a mechanism of self tolerance in hosts that express the activating class I MHC ligand. Using flow cytometry, bone marrow transplant assays, and the in vitro cytotoxicity assay for NK function, we now report that self tolerance in D^d+ mice does not involve deletion or functional anergy of the potentially autoreactive Ly49D⁺ cells. Furthermore, our data supports an alteration (skewing) of the NK cell repertoire, such that most Ly49D⁺ cells from D^d+ mice coexpress a sufficiently calibrated level of self-inhibitory Ly49 receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

All mice were bred and maintained in the Microbiology Colony at the University of Texas Southwestern Medical Center (Dallas, TX). BALB.NK1.1 mice, a gift from Dr. Coffman (DNAX, Palo Alto, CA), are BALB/c mice homozygous for the B6 type NKR-P1C locus. Derivation of these mice has been described (44). Using PCR primers that amplify D6 Mit59 and D6 Mit201, it was determined that the site of crossover in these mice occurred 4–14 cM distal to the Ly49 locus, and thus BALB.NK1.1 mice are also homozygous for the B6 type Ly49 locus. Derivation of the TAP^-/- mutant mice has been described (45). The haplotypes of the various strains used are given in Table I.

The unconjugated 4E5 (anti-Ly49D) and FITC-conjugated 4D11 (anti-Ly49A/G2) mAbs were prepared as described (38, 46). PE conjugation of 4E5 was done by Southern Biotechnical Associates (Birmingham, AL). Biotinylated 4LO3311 (anti-Ly49C) was prepared as described (26, 47). FITC-conjugated JR9-318 (anti-Ly49A) was also used (48). FITC-conjugated PK136 (anti-NK1.1) and biotin-conjugated 5E6 (anti-Ly49C/I) were purchased from PharMingen (San Diego, CA). The 5E6 mAb was derived as previously described (49). The supernatants from hybridomas grown in serum-free medium (HyClone, Logan, UT) were prepared by affinity chromatography using Affigel protein-A agarose (Bio-Rad, Hercules, CA) for mouse 5E6 or protein G-Sepharose 4 Fast Flow (Pharmacia LKB Biotchnology, Piscataway, NJ) for rat 4D11, according to the instructions of the manufacturer. F(ab')₂ reagents were generated. Briefly, the purified mAbs were dialyzed twice against 0.5x PBS buffer. Pilot digestions using pepsin (Sigma, St. Louis, MO) dissolved in 200 mM sodium citrate buffer (pH 3.5) at a 1:40 pepsin to mAb ratio were performed at 37°C for each mAb to determine the optimal length of digestion. F(ab')₂ were then dialyzed against PBS. The efficiency of digestion was checked by 4–20% gradient SDS-PAGE.

Assay for BMC engraftment

BMC engraftment was measured using the splenic 5-iodo-2'-deoxyuridine (IUdR) uptake assay, as described (6). Briefly, BMC donors were killed by asphyxiation with carbon dioxide. The backbones and femurs were placed in RPMI 1640 medium and gently crushed with a mortar and pestle. The cells were filtered through 100-µm nylon cloth, washed, and resuspended in RPMI. A total of 2.5–3.5 x 10⁶ cells in 0.5 ml were injected into each irradiated recipient through the lateral tail vein. All recipients were lethally irradiated (850 cGy) by exposure to ¹³⁷Cs gamma irradiation on the day of BMC transfer (day 0). A total of 1 mg of ammonium sulfate precipitated 5E6 and/or 0.1 mg of ammonium sulfate precipitated 4E5 mAbs were injected i.p. into the recipient mice 2 days before BMC transplant. Then, 5 days later, the mice were given 25 µg of 5-fluoro-2'-deoxyuridine (Sigma, St. Louis, MO) i.p. to supress endogenous thymidine synthesis. Then, 30–60 min later, 0.3 µCi of 5-[¹²⁵I]iodo-2'-deoxyuridine (¹²⁵IUdR) (Amersham Life Science, Arlington Heights, IL) was given i.p. The recipients were killed 2 h later and the spleens removed and soaked overnight in 70% ethanol to solubilize the non-DNA ¹²⁵I. The incorporated radioactivity in the spleens was measured in a gamma counter.

Enrichment of splenic NK cells

Single cell suspensions of splenocytes were prepared aseptically in complete RPMI 1640 (10% FBS, 100 U/ml streptomycin, 100 µg/ml penicillin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 0.1 mM nonessential amino acids) by gently crushing spleens between the frosted edges of two glass slides. The cells were then washed, resuspended at 50 x 10⁶ cells/ml in PBS containing 2% FBS (PBS/FBS), then incubated with 5 µg/ml anti-FcRIII (2.4G2) mAb to block the FcR. After washing, the cells were resuspended at 60–100 x 10⁶ cells/ml in PBS/FBS, then incubated with StemSep murine NK enrichment mixture containing mAbs CD5, CD22, Gr-1, and TER-119 (Stem Cell Technologies, Vancouver, British Colombia, Canada). After washing and resuspending in PBS/FBS at the same concentration, the cells were incubated with StemSep anti-biotin tetramer, then incubated with magnetic colloid. All incubations were performed for 15 min at 4°C. The cells were then filtered onto a PBS-washed StemSep 0.6-inch column placed inside a VarioMACS magnetic field (Miltenyi Biotec, Auburn, CA). The cells collected in the flow-through typically stained 60–90% NK1.1-positive. The cells were then washed and resuspended at 3 x 10⁶ cells/ml in complete DMEM supplemented with 2.25 x 10^-5 M 2-ME and 500 U/ml recombinant human IL-2 (Chiron, Wapole, MA), and cultured overnight in a 24-well plate at 37°C in a 10% CO₂/air mixture.

Cell sorting and generation of effector cells

The NK-enriched cells described above were harvested, washed, then resuspended at 30 x 10⁶ cells/ml in PBS/FBS. The FcR was blocked as described. Without washing, the cells were incubated with biotinylated anti-Ly49C-specific 4LO3311 mAb. After washing, the cells were resuspended and incubated with 1 µg/ml Red 670-conjugated streptavidin (Life Technologies, Gaithersburg, MD). After washing, the cells were incubated with 5 µg/ml biotin-conjugated anti-Ly49C/I (5E6) and PE-conjugated anti-Ly49D (4E5). The cells were then washed and incubated with 1 µg/ml Red 670-conjugated streptavidin and FITC-conjugated anti-NK1.1 (PK136). All incubations were performed for 15 min at 4°C. After washing, cells with forward and side scatter characteristics of lymphocytes were sorted on the FACStar^Plus device (Becton Dickinson, Mountain View, CA). The recovered cells were cultured at 2.5 x 10⁵ cells/ml, 5 x 10⁴ cells/well in 96-well U-bottom plates in complete DMEM supplemented with 2.25 x 10^-5 M 2-ME and 500 U/ml human recombinant IL-2 for 5 days at 37°C in a 10% CO₂/air mixture.

Generation of target cells

Splenocytes were prepared as described and resuspended at 4–6 x 10⁶ cells/ml in complete RPMI 1640 with 6 µg/ml Con A (Sigma). The cells were cultured 2 ml/well in 24-well plates for 2 days at 37°C in a 5% CO₂/air mixture. The cells were then harvested, washed once, resuspended in 2 ml complete RPMI 1640, then layered on top of 4 ml of Ficoll-Hypaque solution (Pharmacia LKB Biotechnology), and centrifuged at room temperature for 20 min at 1300 x g. The buffy coat containing viable lymphoblasts was removed and washed once in complete RPMI 1640 before radiolabeling.

Cytotoxicity assay

A total of 1.5–2 x 10⁶ target Con A lymphoblasts was incubated for 1.5 h at 37°C in a total volume of 0.6 ml RPMI 1640 with 150–250 µCi sodium chromate (⁵¹Cr) (Amersham Life Science). Radiolabeled cells were washed once, resuspended in 5 ml complete RPMI 1640, and incubated an additional 1 h at 37°C. The cells were washed twice, and diluted to 500 targets per 100 µl of media. Effectors at constant 10:1 E:T ratio in a final volume of 100 µl were added first to the wells of 96-well V-bottom plates. An identical volume of targets was added to the appropriate wells. The coincubation was done in triplicate groups at 37°C in a 5% CO₂/air mixture. Effector cells were preincubated with the equivalent of 2 µg/well 4E5 salt cut, 1 µg/well 4D11 F(ab')₂, or 4 µg/well 5E6 F(ab')₂ for 30–60 min at 37°C. After 4 h of incubation, 100 µl of supernatant was removed and ⁵¹Cr radioactivity was measured in a liquid scintillation counter. Specific lysis represented as the mean ± SEM was calculated as follows: Percent specific lysis = ⁵¹Cr cpm [(ER - SR)/(MR - SR)] x 100, where ER is the experimental ⁵¹Cr release in the presence of effector cells, SR is the spontaneous ⁵¹Cr release in the presence of medium, and MR is the maximum ⁵¹Cr release in the presence of 1.0% Triton X-100. Each cytotoxicity assay was performed at least three times.

Flow cytometry analysis

NK enriched splenocytes were prepared as described. On the day of sorting (day 1 of culture), 3 x 10⁵ cells were set aside for cell surface staining analysis. The FcR was blocked as described. Without washing, the cells were incubated with biotinylated anti-Ly49C-specific 4LO3311 mAb. After washing, the cells were resuspended and incubated with 1 µg/ml Red 670-conjugated streptavidin (Life Technologies). After washing, the cells were incubated with PE-conjugated anti-Ly49D (4E5), FITC-conjugated anti-Ly49A/G2 (4D11), and FITC-conjugated anti-Ly49A (JR9–318) for the Ly49D-C-A/G2 three-color stain. For the Ly49D-C/I-A/G2 three-color stain, 5 µg/ml biotin-conjugated anti-Ly49C/I (5E6) was added to the previous step, followed by washing and incubation with 1 µg/ml Red 670-conjugated streptavidin. All incubations were performed for 15 min at 4°C. After washing, cells with forward and side scatter characteristics of lymphocytes were analyzed on the FACScan device (Becton Dickinson). All data refers to Ly49D⁺ gated cells. The percentage of Ly49I⁺C^- cells = (%Ly49C/I⁺) - (%Ly49C⁺). The percentage of Ly49I⁺A/C/G2^- cells = (%Ly49C/I⁺A/G2^-) - (%Ly49C⁺A/G2^-). The percentage of Ly49C/I⁺ cells that lack Ly49A/G2 = (%Ly49C/I⁺A/G2^-)/(%Ly49C/I⁺). The percentage of Ly49C/I^- cells that lack Ly49A/G2 = (%Ly49C/I^-A/G2^-)/(%Ly49C/I^-). The percentage of Ly49C/I⁺A/G2^- cells that express Ly49C = (%Ly49C⁺A/G2^-)/(%Ly49C/I⁺A/G2^-). The percentages shown in the dot plot quadrants are rounded to two significant digits. The percentages used for calculations were done using unrounded values. The data shown represents 15–30 mice each.

Statistics

Statistical calculations have been described (50). The percentage of ¹²⁵IUdR incorporated into each spleen was calculated and converted into log₁₀ values. The geometric means (95% confidence limits) for each group were then calculated. The significance of difference between the geometric means between any two groups was compared using the Vax program UTSTAT provided by the Academic Computing Service of the University of Texas Southwestern Medical Center. Nonparametric analyses were performed.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Murine NK cells express Ly49 receptors that either stimulate or inhibit NK cell function upon interacting with class I ligands. The existence of class I-specific stimulatory receptors can explain some instances of alloreactivity. Their existence also necessitates a mechanism for self tolerance in hosts that express the stimulatory ligand. In this report, we have studied potential mechanisms that ensure maintainance of self tolerance to the function of the H2-D^d-specific stimulatory receptor Ly49D. Our experiments indicate the following: 1) expression of Ly49D does not result in large scale NK cell deletion in a host that expresses D^d, and 2) Ly49D⁺ NK cells from ligand-expressing mice are capable of allorecognition and are not rendered globally anergic. Furthermore, in accordance with the missing self hypothesis, our data suggests that self tolerance is maintained by the coexpression of a sufficient level of self specific inhibitory receptors on Ly49D⁺ NK cells.

H2-D^d-expressing hosts have a normal percentage of Ly49D⁺ NK cells

Because Ly49D stimulates NK function upon interacting with H2-D^d molecules, there should exist a mechanism that ensures self tolerance to Ly49D-mediated activity in mice that express the stimulatory ligand. One potential mechanism could involve the deletion of NK cells that express Ly49D in D^d+ hosts. Alternatively, the level of expression of Ly49D might be reduced in such mice to the point that self ligand-Ly49D interactions are insufficient to produce stimulation of NK activity. To test these possibilities, we stained for expression of NK1.1 and Ly49D (4E5) on freshly isolated NK-enriched splenocytes from three H2 haplotypes of mice, B6 (H2^b/b), [B6 x BALB.NK1.1]F₁ (H2^b/d), and BALB.NK1.1 (H2^d/d) (Fig. 1), all homozygous for the B6-type NK gene complex. All three strains have large Ly49D⁺ NK cell subsets. Of the total NK1.1⁺ cell population, B6 and BALB.NK1.1 mice have 49% and 47% Ly49D⁺ subsets, respectively, while F₁ mice have a 60% Ly49D⁺ subset. All three strains had similar numbers of NK cells per spleen (data not shown). The mean fluorescence intensity of staining with 4E5 was somewhat higher in B6 mice (618) when compared with F₁ (449) or BALB.NK1.1 (369). Whether this difference is functionally important is tested below. Regardless, it can be concluded that large scale deletion of Ly49D⁺ NK cells during ontogeny is not a major mechanism that ensures self tolerance of the NK cell repertoire.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. H2b/b, H2b/d, and H2d/d mice all possess large Ly49D+ NK cell subsets. Splenic NK cells from B6 (A), [B6 x BALB. NK1.1]F1 (B), and BALB.NK1.1 (C) were enriched by magnetic depletion of T cells, B cells, granulocytes, and erythroid cells and were cultured overnight in IL-2. The cells were then stained with FITC-conjugated anti-NK1.1 and PE-conjugated 4E5. The data represent staining of pooled cells from 15 mice each.

Because H2^b/d and H2^d/d mice possess a large Ly49D⁺ subset, we tested the possibility that such cells are rendered functionally anergic in these hosts. If these cells are globally anergic, then H2^d/d hosts would not be expected to reject BMC allografts that express stimulatory ligands for Ly49D. Since the B6 Ly49D receptor has been shown to react with D^d, D^r, and D^sp2 (42), we reasoned that a functional Ly49D⁺ subset from B6 mice would be responsible for the rejection of B10.R40 (K^b, D^sp2) BMC, while an anergic subset from B10.D2 mice would not. As shown in Fig. 2, B10.R40 BMC were rejected by B6 (H2^b/b) and B10.D2 (H2^d/d) recipients. In contrast, B10.R40 BMC grew in TAP^-/- recipients, consistent with the poor ability of NK cells from class I-deficient hosts to mediate BMC rejection (27, 51, 52). To assess the potential role that Ly49D⁺ and Ly49C/I⁺ cells play in the rejection of D^sp2+ BMC, we assayed the effect that 4E5 (anti-Ly49D) and/or 5E6 (anti-Ly49C/I) depletion had on the outcomes of BMC rejection. Animals treated with 4E5 had no detectable splenic Ly49D⁺ cells, and those treated with 5E6 had no detectable Ly49C/I⁺ cells (data not shown). Depletion of the Ly49D⁺ subset with 4E5 mAbs reversed the ability of both B6 and B10.D2 recipients to reject the allogeneic BMC graft, demonstrating that regardless of host expression of H2-D^d, the Ly49D⁺ subset is capable of rejecting D^sp2+ BMC grafts (Fig. 2). Furthermore, depletion of B10.D2 5E6⁺ cells has no effect on the rejection, demonstrating that the Ly49D⁺C/I^- subset that remains after such depletion is fully capable of allorecognition of the B10.R40 graft (Fig. 2B). Thus, although the H2^d/d-derived Ly49D subset is self tolerant, it is fully capable of mediating the rejection of allogeneic BMC grafts that express a non-D^d ligand for the Ly49D receptor (42).

View larger version (14K): [in this window] [in a new window]   FIGURE 2. The Ly49D+ subset from H2b/b and H2d/d mice is responsible for the rejection of H2Dsp2-expressing BMC grafts. A total of 3.5 x 106 BMC from B10.R40 donors were infused into lethally irradiated TAP-/- and B6 (A) or B10.D2 (B) recipients. Some recipients were injected with 100 µg of 4E5 (anti-D) and/or 1.0 mg or 5E6 (anti-C/I) salt-cut mAbs i.p. 24 h before transplantation of donor BMC. Then, 5 days after transplantation, splenic uptake (%) of 125IUdR was measured. Each group contained at least four mice. , Individual recipient mice; •, geometric means for each group. *, Geometric mean values were statistically significant from other groups, but not from each other, p < 0.05.

The previous data suggest that self tolerance results in specific unresponsiveness of the Ly49D subset to syngeneic cells, while allowing for responsiveness to allogeneic cells. This raises the possibility that target cells that express D^d might be resistant to Ly49D-mediated rejection by all D^d+ hosts. As hypothesized by Raziuddin et al. (41), development in a D^d+ environment might result in a specific functional alteration in the Ly49D receptor such that it can only stimulate NK function upon interacting with allogeneic class I ligands (such as D^sp2). If this were the case, the ability of H2^b/d F₁ NK cells to reject H2^d/d parental BMC would be mediated through some non-Ly49D-mediated signal (6). In fact, a previous study showed that Abs to NKR-P1C slightly reduced the lysis of H2^d/d targets by H2^b/d F₁ NK cells (4). To test the functional role that F₁ Ly49D⁺ NK cells play in hybrid resistance, we compared the ability of B6 and [B6 x BALB.NK1.1] F₁ NK subsets to reject H2^d/d BMC. Both B6 and F₁ mice reject H2^d/d BMC (Fig. 3). Administration of anti-Ly49D 4E5 reverses the rejection of BALB/c (H2^d/d) BMC grafts by both hosts (Fig. 3, A and B). This reproduces earlier data demonstrating that Ly49D⁺ B6 NK cells are responsible for the rejection of H2^d/d BMC grafts (41). Additionally, it can be concluded that Ly49D⁺ NK cells from D^d-expressing F₁ mice are functionally capable of rejecting D^d-expressing grafts (Fig. 3B). In contrast, mAbs to Ly49C/I (5E6) reverse hybrid resistance but not rejection of H2^d/d BMC by B6 hosts. Thus, the Ly49D⁺C/I^- subset from B6 mice, which remains after 5E6 depletion, is capable of rejecting the H2^d/d BMC. In contrast, any F₁ Ly49D⁺C/I^- NK subset that remains after 5E6 depletion, is incapable of rejecting H2^d/d BMC. It follows that the F₁ Ly49D⁺C/I⁺ NK subset is responsible for hybrid resistance to H2^d/d BMC grafts. This conclusion can explain data from earlier reports that demonstrated the 5E6+ F₁ NK subset mediates hybrid resistance to H2^d/d BMC grafts (41, 49). Regardless, it is clear that host expression of D^d is not sufficient to render Ly49D⁺ cells tolerant to all D^d-expressing BMC grafts. At this point, it could still be argued that Ly49D⁺ cells kill by a mechanism other than Ly49D-D^d stimulatory interactions. This is directly addressed in the next section.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Both the Ly49D+ and Ly49C/I+ subsets from H2b/d F1 mice are responsible for rejecting H2d/d parental BMC grafts. A total of 3.5 x 106 BMC from BALB/c donors were infused into irradiated BALB/c and B6 (A) or [B6 x BALB. NK1.1]F1 (B) recipients. Some recipients were injected with 4E5 (anti-Ly49D) and/or 5E6 (anti-Ly49C/I) mAbs i.p. (see Fig. 2). Then, 5 days after transplantation, splenic uptake (%) of 125IUdR was measured. Each group contained at least four mice. , Individual recipient mice; •, geometric means for each group. *, Geometric mean values were statistically significant from other groups, but not from each other, p < 0.05.

The Ly49D⁺C/I⁺ subset from B6 and F₁ but not BALB.NK1.1 mice can lyse H2^d/d Con A lymphoblasts in vitro

The previous transplant data demonstrates that the F₁ Ly49D⁺C/I⁺ subset is not tolerant to H2^d/d BMC, while the corresponding BALB.NK1.1 subset is tolerant. To identify the role that the Ly49D receptor plays in the recognition of H2^d/d cells by this subset from these mice, we performed in vitro cytotoxicity assays, using sorted IL-2 activated Ly49D⁺C/I⁺ NK cells as effectors in the presence or absence of anti-Ly49 reagents against a panel of ⁵¹Cr-labeled Con A-stimulated lymphoblasts as targets (Fig. 4). Ly49D⁺C/I⁺ effector subsets derived from any of the three haplotypes lysed the K^b+ targets (B6, F₁, or B10.R40) poorly in the absence of blocking reagent. Preincubation of the NK cells with anti-Ly49C/I 5E6 F(ab')₂ fragments increased the lysis of these targets. This supports the fact that Ly49C/I interactions with K^b result in inhibition of NK cells function (21). It furthermore suggests that this inhibitory interaction dominates positive signaling from Ly49D-D^d interactions, resulting in self tolerance to F₁ targets. In contrast, Ly49D⁺C/I⁺ B6 and F₁ cells efficiently lysed B6.C (H2^d/d) targets (Fig. 4, A and B). Lysis of this target was significantly reduced when the effectors were preincubated with anti-Ly49D 4E5 mAbs, suggesting that, in these mice, the interactions between Ly49D and H2^d/d class I molecules result in activation of NK cell function. 4E5 mAb preincubation did not reduce the lysis of TAP^-/- targets, demonstrating that such treatment was not toxic to the Ly49D⁺ cells (data not shown). Furthermore, preincubation of the B6 and F₁ effectors with 5E6 F(ab')₂ fragments only slightly increased the lysis of the B6.C target. Thus, any Ly49C/I-H2^d/d inhibitory signaling that is present either does not fully dominate Ly49D-mediated signaling or occurs only in some of the cells in this subset. Therefore, we conclude that Ly49D⁺C/I⁺ NK cells from B6 mice lyse or reject H2^d/d cells due to the presence of Ly49D-D^d stimulatory signaling in the absence of strong Ly49C/I-H2^d/d inhibitory signaling. Strong Ly49C/I-K^b inhibitory signaling ensures self tolerance of NK cells from these mice.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. The Ly49D+C/I+ subset from BALB. NK1.1 but not B6 or [B6 x BALB. NK1.1]F1 mice is tolerant to H2d/d Con A lymphoblasts in vitro. Ly49D+C/I+ LAKs from B6 (A), [B6 x BALB. NK1.1]F1 (B), or BALB.NK1.1 (C) were generated as described and were coincubated at a 10:1 E:T ratio with a panel of 51Cr-labeled Con A blasts in the presence or absence of 2 µg/well of blocking anti-Ly49D 4E5 mAbs, 4 µg/well anti-Ly49C/I 5E6 F(ab')2, or 1 µg/well anti-Ly49G2 4D11 F(ab')2. Preincubation with blocking reagent was performed 30 min before the addition of target cells.

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Pattern of coexpression on Ly49D+ splenic NK cells reveals both alterations in inhibitory receptor repertoire and in calibration of inhibitory receptor expression. Splenic NK cells from B6 (A, D, and G), [B6 x BALB.NK1.1]F1 (B, E, and H), and BALB. NK1.1 (C, F, and I) were enriched by magnetic depletion of T cells, B cells, granulocytes, and erythroid cells and were cultured overnight in IL-2. The cells were then stained with PE-conjugated anti-Ly49D (4E5), FITC-conjugated anti-Ly49G2 (4D11), and anti-Ly49A (JR9–318), and biotin-conjugated anti-Ly49C (4LO3311) alone (D–F) or along with anti-Ly49C/I (5E6) (A–C), followed by RED670-conjugated streptavidin. The dot plots represent gated Ly49D+ cells. Data for pie charts (G–I) are derived from dot plots. The percentage of (I+C-)A/G2- cells was derived by subtracting the (C+)A/G2- percentage from the (C/I+)A/G2- percentage. All data shown has been rounded to two significant digits. Calculations of data derived from the dot plots and pie charts are described in Materials and Methods.

The Ly49D⁺C/I^- subset from B6 but not F₁ or BALB.NK1.1 mice can lyse H2^d/d Con A lymphoblasts in vitro

The previous transplant data suggested that the Ly49D⁺C/I^- NK cells from B6 but not F₁ or BALB.NK1.1 hosts are capable of rejecting H2^d/d BMC. To test the function of the Ly49D receptor in these subsets, we evaluated the lytic potential of sorted IL-2 activated Ly49D⁺C/I^- subsets against a panel of target cell lymphoblasts (Fig. 5) in the cytotoxicity assay. All effectors efficiently lysed B10.R40 targets, and, in all cases, lysis of the B10.R40 target was significantly reduced in the presence of anti-Ly49D reagents. Thus, in the absence of Ly49C/I-K^b inhibitory interactions, Ly49D-D^sp2 stimulatory interactions result in activation of NK-mediated lysis. Only the D^d-expressing F₁ and BALB.NK1.1 NK cells were capable of lysing H2^b/b target cells, consistent with the lack of Ly49C/I expressed by these NK cells (Fig. 5, B and C). Anti-Ly49D preincubation did not, however, significantly reduce the lysis of the B6 target, suggesting that some non-Ly49D-mediated mechanism accounted for the lysis. It is likely that a tolerance mechanism operating in B6 mice prevents the autoreactivity of this subset. Consistent with the previous transplant data, Ly49D⁺C/I^- cells from the D^d-expressing F₁ and BALB.NK1.1 hosts were unable to lyse D^d+ F₁ or B6.C target cells in the absence of blocking Abs, unlike the analogous subset isolated from D^d- B6 mice (Fig. 5C). However, preincubation with anti-Ly49A/G2 F(ab')₂ significantly increased the lysis of the D^d-expressing targets by this subset from all three strains. This suggests that a significant proportion of these cells are tolerant to D^d-expressing target cells due to coexpression of the D^d-specific inhibitory receptors, Ly49A/G2. This possibility is tested below. Consistent with previous results (42), when a cocktail of 4D11 and 4E5 was added to these effectors, lysis of the H2^d/d target was reduced significantly but not completely abolished (data not shown), suggesting that other unknown activating receptors can function in this system.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. The Ly49D+C/I- subset from [B6 x BALB. NK1.1]F1 and BALB.NK1.1, but not B6, mice is tolerant to H2d/d Con A lymphoblasts in vitro. Ly49D+C/I- LAKs from B6 (A), [B6 x BALB. NK1.1]F1 (B), or BALB. NK1.1 (C) were generated as described and were coincubated at a 10:1 E:T ratio with a panel of 51Cr-labeled Con A blasts in the presence or absence of 2 µg/well of blocking anti-Ly49D 4E5 mAbs or 1 µg/well anti-Ly49G2 4D11 F(ab')2. Preincubation with blocking reagent was performed 30 min before the addition of target cells.

The class I specificity of a particular NK cell subset depends, at least in part, on the array of class I-specific Ly49 receptors expressed by cells within that subset. Because Ly49 receptor inhibitory signaling often dominates NK cell activity (42, 58, 59), functional differences of a particular Ly49D⁺ NK subset from H2^b/b, H2^b/d, and H2^d/d mice might reflect differences in coexpression of dominantly functioning inhibitory Ly49 receptors. We predicted that these functional differences result from a mechanism that ensures self tolerance of the host NK cell repertoire. If coexpression of self-specific inhibitory Ly49 receptors contributes to self tolerance, we expected to see differences among the three haplotypes in receptor coexpression patterns on Ly49D⁺ cells. To test this, we performed three-color staining of NK-enriched fresh splenocytes from all three haplotypes to examine coexpression of D^d inhibitory receptors (Ly49A and G2) and H2^b/b inhibitory receptors (Ly49C and I or Ly49C alone) on cells that expressed the D^d stimulatory receptor (Ly49D) (Fig. 6). Calculations of cell subset percentages are given in Materials and Methods.

We will first discuss self tolerance of the B6 Ly49D⁺ subset. Of significant importance is the fact that there is no evidence that B6 cells express a stimulatory ligand for Ly49D. Ly49D-depleted F₁ mice are fully capable of rejecting B6 BMC grafts (Ref. 41 and data not shown). Also, in no case does anti-Ly49D block the lysis of B6 targets (Figs. 4 and 5). Therefore, expression of Ly49D in a B6 environment may have no bearing on self tolerance. Both Ly49C and Ly49I, but not Ly49A or Ly49G2, NK cell receptors from B6 mice interact with self H2^b class I Ags. Approximately 79% of B6 Ly49D⁺ cells coexpress Ly49C/I and, as a result, are self tolerant (Fig. 6, A and G). The remaining 21%, which remain after 5E6 depletion, are fully capable of rejecting H2^d/d BMC grafts in vivo (Fig. 3A), and lysing H2^d/d- and D^sp2-expressing targets in vitro (Fig. 5A); therefore, they are not anergic. They may be self tolerant because 1) they lack a self-specific stimulatory receptor, or 2) they coexpress unknown self-specific inhibitory receptors. The fact that self-tolerant Ly49D⁺A/C/G2/I^- B6 (K^bD^b) cells efficiently lysed D8 (K^bD^b/d) targets in vitro suggests that these cells do not express a self-specific inhibitory receptor capable of dominating the Ly49D-D^d stimulatory interaction (42). Thus, these cells may be self tolerant due to a lack of expression on a self-specific stimulatory receptor.

In contrast to B6 mice, F₁ mice express a stimulatory ligand for Ly49D. Also in contrast to B6 mice, self tolerance of F₁ Ly49D⁺ cells can be achieved through coexpression of Ly49A, C, G2, or I. A total of 97.8% of F₁ Ly49D⁺ cells coexpress at least one of these self-specific inhibitory receptors (Fig. 6, B and H). The remaining 2.2% are either anergic, or are self tolerant, due to coexpression of an unknown inhibitory receptor. In vivo and in vitro data demonstrated that the Ly49D⁺C/I⁺ F₁ subset, which is self tolerant due to dominant Ly49C/I-K^b inhibitory interactions, is responsible for the hybrid resistance to H2^d/d cells (Figs. 3B and 4B). The alloreactivity of this subset to the H2^d/d parent is likely due to the fact that 36.8% of these F₁ NK cells lack coexpression of Ly49A and G2 (derived from Fig. 6H). On the other hand, the F₁ Ly49D⁺C/I^- subset, which remains after 5E6 depletion, is incapable of rejecting H2^d/d BMC (Fig. 3B). This NK subset could not lyse D^d-expressing target cells, unless Ly49A/G2 receptors were blocked with 4D11 F(ab')₂ fragments, and, thus, this F₁ subset is also self tolerant (Fig. 5B). Consistent with the functional data is the fact that only 10.2% of Ly49D⁺C/I^- cells lacked coexpression of Ly49A and Ly49G2 (derived from Fig. 6H). Thus, self tolerance of the F₁ Ly49D⁺C/I^- cells is achieved, in large part, by coexpression of D^d-specific inhibitory receptors.

Unlike B6 or F₁ Ly49C/I⁺ subsets, BALB.NK1.1 (H2^d/d) Ly49C/I-H2^d/d interactions can dominantly inhibit Ly49D-mediated stimulation. How is this possible? The staining data reveal two informative trends that pertain to tolerance to H2^d/d target cells. Within the Ly49D⁺C/I⁺ subset, only the Ly49A/G2^- cells are potentially autoractive in an H2^d/d host. In BALB.NK1.1 mice, 81.8% (compared with 71.0% of F₁ and 56.5% of B6) of these Ly49D⁺C/I⁺A/G2^- cells express Ly49C (derived from Fig. 6, G–I). Furthermore, the mean fluorescence intensity of BALB.NK1.1 Ly49C is 216 compared with 66 for F₁ and 50 for B6 cells (Fig. 6, D–F), consistent with previous reports that development in a K^b+ environment results in a drastic down calibration of Ly49C (26, 27, 28, 29). Also consistent with previous reports, development in an H2^d/d environment results in a less severe down calibration of Ly49C (26). Thus, we propose that the increased frequency and staining intensity of the Ly49C receptor is primarily responsible for the self tolerance of the BALB.NK1.1 Ly49C/I⁺A/G2^- subset. Therefore, 94.9% of the BALB.NK1.1 Ly49D⁺ subset maintains self tolerance by virtue of coexpression of Ly49A, C, or G2 (Fig. 6I); 3.6% coexpress Ly49I only. These latter cells may coexpress another self-specific inhibitory receptor. Alternatively, Ly49I may, in some instances, be capable of functionally interacting with H2^d/d Ags. We are currently testing these possibilities in vitro. Finally, only 1.5% of BALB.NK1.1 Ly49D⁺ cells lack coexpression of Ly49A, C, G2 and/or I. Again, these latter cells may be self tolerant due to coexpression of other receptors, or are anergic.

Thus, we conclude that self tolerance of Ly49D⁺ cells in D^d+ mice is predominantly maintained by the coexpression of a sufficient level of self-specific inhibitory Ly49 receptors. At the same time, the alloreactive potential of self-tolerant NK cells is maintained by several mechanisms. 1) Stimulatory class I receptors interact with a different pool of ligands than inhibitory class I receptors do (Fig. 7A). For example, Ly49A/G2 coexpression renders an NK cell from a D^d+ mouse self tolerant, but does not inhibit reactivity against a D^sp2+ target. 2) The host expresses a different pool of class I Ags than the target (Fig. 7B). Thus, Ly49I coexpression prevents autoreactivity of an H2^b/d-derived Ly49D⁺ NK cell, but may not protect an H2^d/d (K^b-, D^d+) target from lysis. 3) The host may down calibrate a self-specific inhibitory receptor such that it can protect self targets but not other targets from lysis or rejection by Ly49D⁺ NK cells (Fig. 7C). For example, F₁ Ly49D⁺C^dim NK cells have a sufficient amount of Ly49C expression to interact with self K^b Ags, but an insufficient level to interact with H2^d/d parental Ags. 4) Some stimulatory class I receptors may only interact with alloantigens (Fig. 7D). Thus, B6 Ly49D⁺ cells that do not coexpress any self-specific inhibitory Ly49 receptor probably lack a self-specific stimulatory receptor. Hematopoietic cells whose class I quality or content are altered due to viral infection, transformation, or mutation may become susceptible to a particular self-tolerant NK subset by: 1) losing inhibitory class I motifs, or 2) gaining stimulatory class I motifs. Either occurrence may result in susceptibility to particular subsets of host NK cells. Specific NK cell subsets may be sensitively poised to detect certain alterations in class I content and quality during cellular transformation. The existence of many self-tolerant NK cell subsets that express different combinations of stimulatory and inhibitory receptors may allow for the sensitive detection of a great variety of significant alterations in class I molecules on autologous cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Coexpression of self-specific inhibitory Ly49 receptors ensures the self tolerance of, but does not preclude the alloreactivity of, Ly49D+ NK cells. A, In an H2d/d mouse, Ly49G2 protects Dd+ but not Dsp2+ targets from Ly49D-mediated stimulation. B, In an H2b/d mouse, Ly49I protects Kb+ but not Kb- targets from Ly49D-mediated stimulation. C, In an H2b/d mouse, Ly49Cdim protects Kb+ but not Dd+Kb- targets. D, In an H2b/b mouse, Ly49D does not react with self class I Ags, and potentially can be self tolerant without coexpression of inhibitory Ly49 receptors.

	Acknowledgments

 
We thank Drs. Llewellyn H. Mason, Noelle S. Williams, and John D. Schatzle for review of the manuscript, Beni Stewart for assistance with figures, Angie Mobley for expert assistance with the flow cytometry, Deming Zhou for preparation of F(ab')₂ reagents, Mesha Austin for PCR typing of the BALB.NK1.1 mice, and Silvio and Maria Peña for excellent breeding and maintenance of mice.

	Footnotes

 
¹ This work was supported by Grants AI38938, CA36921, AI25401, and CA09082 from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Michael Bennett, Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235-9072. E-mail address:

³ Abbreviations used in this paper: BMC, bone marrow cell; ITIM, immunoreceptor tyrosine-based inhibitory motif; ¹²⁵IUdR, 5-[¹²⁵I]iodo-2'-deoxyuridine.

⁴ J. Liu, M. A. Morris, P. Nguyen, T. George, W. C. Lai, J. D. Schatzle, V. Kumar, and M. Bennett. Ly49I NK cell receptor transgene inhibition of H2^b mouse bone marrow transplant rejection. Submitted for publication.

Received for publication March 24, 1999. Accepted for publication June 7, 1999.

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