The class Ib molecule Qa-1b binds the class Ia leader peptide, Qdm, which reacts with CD94/NKG2R on NK cells. We have generated a gene that encodes the Qdm peptide covalently attached to β2-microglobulin (β2M) by a flexible linker (Qa-1 determinant modifier (Qdm)-β2M). When this construct is expressed in TAP-2 or β2M cells, it allows for the expression of a Qdm-β2M protein that associates with Qa-1b to generate the Qdm epitope, as detected by Qdm/Qa-1b-specific CTL. To test the biological significance of expression of this engineered molecule, we injected TAP-2 RMAS-Qdm-β2M cells into C57BL/6 mice and measured their NK cell-mediated clearance from the lungs at 2 h. RMAS cells transfected with Qdm-β2M were resistant to lung clearance, similar to RMA cells or RMAS cells in anti-asialo-GM1-treated mice, while untransfected or β2M-transfected RMAS cells were rapidly cleared. Further, pulsing RMAS cells with either Qdm, a Kb-, or Db-binding peptide showed equivalent protection from clearance, indicating that a single class Ia or Ib molecule can afford complete protection from NK cells in this system. In contrast, injection of RMAS cells into DBA/2 animals, which express low levels of receptors for Qdm/Qa-1b, resulted in protection from lung clearance if pulsed with a Kb- or Db-binding peptide, but not the Qa-1b-binding peptide, Qdm.

Natural killer cells express both activating and inhibitory receptors that regulate their ability to interact with other cells in their environment (1, 2, 3). Many inhibitory receptors have been identified in both mouse and human that interact with self class I molecules, and as a result of this interaction, NK cells are inhibited from killing target cells both in vitro and in vivo. Murine NK cells express two known families of inhibitory receptors that use class I molecules as their ligands, Ly49, and CD94/NKG2A (4, 5). Both of these receptors are C-type lectins with cytoplasmic domains that contain immunoreceptor tyrosine-based inhibition motifs. The cross-linking of such receptors results in phosphorylation of these immunoreceptor tyrosine-based inhibition motifs with their subsequent interaction with SHP-1 and -2 phosphatases, which presumably dephosphorylate activating substrates and prevent the activation of NK cells (6).

The class Ib molecule Qa-1b has been shown to bind a nonamer peptide derived from the leader of class Ia molecules (7). This peptide is referred to as the Qa-1 determinant modifier (Qdm)3 and binds to Qa-1b with very high affinity (8). Qdm/Qa-1b binds to CD94/NKG2AR on NK cells and, as a result, mediates an inhibitory signal (9, 10). Therefore, NK cells can receive inhibitory signals from class Ia molecules directly by binding to Ly-49 or killer cell inhibitory receptors in mouse or human, respectively, or indirectly via presenting their leader peptide in the groove of Qa-1b to CD94/NKG2A. Although Qa-1b binds the Qdm peptide with high affinity, it is also known to bind other peptides (Ref. 11 and data not shown). Thus, Qa-1b bound to peptides other than Qdm may play a role in inhibiting NK cells either by interacting with CD94/NKG2A or unknown receptors. To this end, we have generated a β2M gene construct that has the Qdm peptide covalently attached through a flexible linker. Upon transfection, this construct allows for expression of Qdm/Qa-1b on cells defective in either TAP or β2M. We have used a TAP transfectant to assess the ability of such cells to be protected from NK-mediated lung clearance in vivo. Since CD94/NKG2A as well as Ly49 receptors for class I ligands are expressed only on a subset of NK cells, we compared the ability of Qdm/Qa-1b, Kb, and Db to mediate protection from in vivo NK-mediated lung clearance.

C57BL/6 (B6) and DBA/2 animals were bred and maintained in our animal colony at University of Texas Southwestern Medical Center (Dallas, TX).

RMA/S-Qa1b are Qa1b transfectants that express a higher level of Qa-1b than wild-type RMAS cells as determined by Western blot analysis with an anti-Qa-1b antiserum (data not shown). KJ-29 (12) is a human renal carcinoma cell line that lacks β2M expression. Other tumor lines have been previously described (13). CTL clone 3C9 recognizes the Qdm peptide (AMAPRTLLL) associated with Qa-1b (7).

Mouse β2M cDNA from BALB/c mice was used as a template to generate a β2M molecule containing a flexible linker attached to the Qdm nonamer peptide (Qdm-β2M). Our approach is a modification of that previously described by Uger and Barber (14). Qdm-β2M was generated by overlapping PCR (Fig. 1). To generate the 5′ end of the construct (template 1) containing the β2M leader, Qdm, and flexible linker, an upstream primer (primer 1) 5′-CTAAGCTTGCCACCATGGCTCGCTCGGTGACCCTAGTC-3′ (which includes a HindIII restriction site and a Kozak sequence), that hybridizes to the β2M signal sequence was used. There were five downstream primers as follows: 5′-GCGCGGAGCCATCGCAGCATACAAGCCGGTCAGTGAGAC-3′, 5′-TCCCAGGAGCAGCGTGCGCGGCGCCATCGCAGCATACAAG-3′, 5′-TCCTCCAGATCCTCCTCCCAGGAGCAGCGTGCGCGGAGC-3′, 5′-TCCTCCTCCAGATCCTCCTCCAGATCCTCCTCCCAGGAG-3′, 5′-GGTTTTCTGGATAGATCCTCCTCCAGATCCTCCTCCAG-3′ (primers 2–6, respectively), which contain the sequence encoding the Qdm (AMAPRTLLL) epitope and a glycine-serine linker (GGGS)3. The resulting PCR products were then used as templates for each subsequent step. To generate the 3′ end of the construct containing the mature β2M protein (template 2), wild-type β2M was amplified using primer 7, 5′-TCTGGAGGAGGATCTATCAAAACCCCTCAAATTC-3′, which hybridizes to β2M and a portion of the linker, and one downstream primer, 5′-CGTTCTAGATCACATGTCTCGATCCCAGTAGACGGTCTTG-3′ (primer 8), containing an XbaI restriction site. For the complete construct, we used primer 1 and 8 together with the two templates to generate the full-length PCR product. The Qdm-β2M product was cloned into the pcDNA 3.1(+) vector containing the neomycin resistance gene.

FIGURE 1.

Generation of the Qdm-linker-β2M and Qdm-linker-β2M-HIS gene constructs. See text for details.

FIGURE 1.

Generation of the Qdm-linker-β2M and Qdm-linker-β2M-HIS gene constructs. See text for details.

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To insert a (HIS)6 tag at the C-terminal end of the construct, we amplified Qdm-β2M with two PCR using the upstream primer (primer 1) and two new downstream primers (5′-ATGATGATGCATGTCTCGATCCCAGTAGAC-3′ and 5′-TCAATGATGATGATGATGATGCATGTATCG-3′; primers 9 and 10, respectively), which hybridize to the C-terminal end of β2M and contain additional sequences encoding the (HIS)6 tag and stop codon. These primers allow for the extension of the 3′ end of the gene to include the (HIS)6 tag and a stop codon.

Three micrograms of β2M or the Qdm-β2M plasmid were transfected into 106 RMAS, 2 × 105 Ltk, and 2 × 105 KJ-29 cells with Fugene 6 (Roche Diagnostic Systems, Summerville, NJ) and LipofectAmine reagent (Life Technologies, Rockville, MD). After incubation for 5 h at 37 C in a CO2 incubator, 5 ml of medium (with 20% FBS, but no antibacterial agents) was added, and the transfectants were cultured for 2 days before being transferred to G418 selection medium. For cells that contained the neomycin resistance gene (i.e., RMA/S-Qa1b (1 × 106) and L-Qa-1b (2 × 105) cells), the gene constructs were cotransfected with the Hygromycin-B-phosphotransferase gene at a 10:1 ratio. The transfectants were cultured with the same complete growth medium for 48 h and then transferred to hygromycin selection medium.

Cells (5 × 105) were lysed on ice with 0.5 ml of lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 ml iodoacetamide, 1 mM PMSF, 0.1 U/ml trypsin inhibitor aprotinin) for 30 min. The postnuclear supernatants were incubated for 1 h at 4 C with nickel-nitrilotriacetic acid agarose (Qiagen, Chatsworth, CA). After two washes with PBS, 150 μl 20 mM imidazole was added at room temperature for 5 min, the beads were spun, resuspended in 50 μl 150 mM imidazole, and recentrifuged. The supernatant was used as the sample to run 10% SDS-PAGE gels and transferred to nitrocellulose (Amersham Pharmacia Biotech, Piscataway, NJ). The filters were probed with an anti-HIS mAb (Sigma, St. Louis, MO) at a 1:1000 dilution and detected with anti-mouse HRP conjugate (Amersham, Arlington Heights, IL) at a 1:4000 dilution using the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech).

The Qa1b-specific CTL clone 3C9 is specific for the Qdm peptide bound to Qa-1b (7). In experiments involving peptide pulsing, target cells were kept at room temperature overnight. Recombinant vaccinia virus (rVV) containing the Qa1b gene was used to infect human KJ-29 cells (which do not encode Qa-1b) 2 h before being labeled with 51Cr. Labeled target cells (104) were dispensed into 96-well plates and 100 ng/well Qdm peptide was added where indicated at room temperature 30 min before the addition of effector cells.

RMA/S-Qa1b and their transfectants (106) were incubated at room temperature overnight and pulsed with the Qa1b-binding peptide Qdm (AMAPRTLLL), Kb-binding peptide (SIINFEKL), and the Db-binding peptide (ASNENMETM) at a concentration of 1 μg/ml. Subsequently, the cells were incubated on ice for 1 h with anti-Qa1b mAb 910 (conjugated with PE), anti-Kb, and anti-Db (conjugated with FITC) Ab (PharMingen, San Diego, CA) at a 1:100 dilution with FACS buffer. RMA/S-Qa1b and the transfectants (106) were also stained with an anti-HIS tag (C-terminal) mAb (primary Ab) and then an anti-mouse Ig (PE conjugate). Cells were washed three times and resuspended in 0.5 ml FACS buffer. Samples were analyzed on a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA).

We used an assay described by Hackett et al. (15). Briefly, RMA, RMA/S-Qa1b, and their transfectants were incubated with different peptides at a concentration of 1 μg/ml at room temperature overnight. The cells were then incubated in RPMI 1640 with 25 μg/ml 5-fluordeoxyuridine (Sigma) for 15 min, and then 30 μCi 5-iodo-2′deoxyuridine-125I (125IUdR; ICN Pharmaceuticals, Costa Mesa, CA) was added and the cells were incubated an additional 60 min at 37°C in 5% CO2. The cells were washed three times in complete medium before injection. In some experiments, B6 mice were treated with 20 μl anti-asialo GM1 (Wako Pure Chemical, Osaka, Japan) i.p. 1 day before labeled cells were injected. 125IUdR-labeled cells (1 × 106; 200 μl) were injected into the lateral tail vein of individual mice. At 2–2.5 h after injection, the mice were sacrificed, the lungs were removed, and the 125I radioactivity was counted.

We generated a β2M gene containing the Qdm peptide covalently attached through a flexible linker following an approach described by Uger and Barber (14). The construct consisted of the β2M leader, a 12-aa flexible linker upstream of the 27-bp segment encoding Qdm followed by the β2M coding sequence. This construct was transfected into several cell lines including RMAS, RMAS-Qa-1b, T2-Qa-1b, Ltk, L-Qa-1b, and a β-2M cell line, KJ29. Control transfections consisted of the β2M gene lacking the Qdm epitope. All cell lines were initially tested for successful transfection by PCR using Qdm-β2M-specific primers (data not shown). We wished to demonstrate that the transfected cell lines contained two β2M molecules, one representing native β2M, the other the Qdm-β2M protein. Because the size of Qdm-β2M and native β2M molecules is similar, it was not easy to distinguish the two species by Western blot analysis using the anti-β2M sera available (data not shown). Therefore, we inserted a (HIS)6 tag at the C terminus of the Qdm-β2M and β2M constructs so that they could readily be detected using an anti-HIS mAb. The data in Fig. 2 clearly show that both RMAS and RMAS-Qa-1b cells transfected with the Qdm-β2M-(HIS)6 construct expresses a protein detected by Western blot that migrates slower than wild-type β2M-(HIS)6 (lanes 3 vs 2 and 6 vs 5), consistent with the former having the Qdm linker attached. Thus, this indicates that the Qdm epitope remains associated with β2M and is not postranslationally cleaved by host cell enzymatic activity.

FIGURE 2.

Western blot analysis of anti-HIS-binding proteins. RMAS or RMAS-Qa-1b cells were transfected with β2M-(HIS)6 or Qdm-β2M-(HIS)6. Lysates were eluted from Ni-nitrilotriacetic acid agarose, run on a 10% SDS-PAGE gel, and transferred to nitrocellulose before blotting with anti-HIS. Lanes 1 and 4, untransfected; lanes 2 and 5, β2M-(HIS)6; lanes 3 and 6, Qdm-β2M-(HIS)6.

FIGURE 2.

Western blot analysis of anti-HIS-binding proteins. RMAS or RMAS-Qa-1b cells were transfected with β2M-(HIS)6 or Qdm-β2M-(HIS)6. Lysates were eluted from Ni-nitrilotriacetic acid agarose, run on a 10% SDS-PAGE gel, and transferred to nitrocellulose before blotting with anti-HIS. Lanes 1 and 4, untransfected; lanes 2 and 5, β2M-(HIS)6; lanes 3 and 6, Qdm-β2M-(HIS)6.

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The above data show that a Qdm-β2M construct can be expressed in several cell types, but does not demonstrate that the Qdm peptide is associated with Qa-1b. Therefore, we used a CTL clone that is specific for the Qdm peptide bound to Qa-1b to determine whether this construct, when transfected into cells, allowed for Qa-1b presentation of the Qdm epitope. RMAS-Qa-1b cells should not express Qdm/Qa-1b complexes on their cell surface because the presentation of the Qdm peptide is TAP dependent (16). As expected, RMAS-Qa-1b cells are not recognized by Qdm-Qa-1b-specific CTL clone 3C9. Nor does transfection of RMAS-Qa-1b cells with the β2M (control) construct allow for this clone to recognize such target cells (Fig. 3,a). However, transfection of RMAS-Qa-1b cells with Qdm-β2M does allow for efficient recognition. In fact, the lysis of this transfectant is greater than that seen when RMAS-Qa-1b cells are pulsed with an optimal dose of the Qdm peptide. Pulsing target cells transfected with Qdm-β2M with the Qdm peptide allows for additional lysis. This result is expected because pulsing RMAS-Qa-1b cells with the Qdm peptide should allow for presentation of the Qdm/Qa-1b epitope by Qa-1b molecules that have associated with endogenous β2M. A similar result is observed with L-Qa-1b cells that do not express the Qdm epitope. Transfection with Qdm-β2M allows for recognition by Qdm-Qa-1b-specific CTL (Fig. 3 b).

FIGURE 3.

Ability of Qdm/Qa-1b-specific CTL clone 3C9 to recognize TAP-2 RMAS-Qa-1b transfectants. a, RMAS-Qa-1b cells were transfected with β2M or Qdm-β2M and tested for their sensitivity to lysis by clone 3C9. Some of the targets were incubated overnight at room temperature and pulsed with 100 ng Qdm before the CTL assay. b, L-Qa-1b cells were transfected with Qdm-β2M and tested as in a.

FIGURE 3.

Ability of Qdm/Qa-1b-specific CTL clone 3C9 to recognize TAP-2 RMAS-Qa-1b transfectants. a, RMAS-Qa-1b cells were transfected with β2M or Qdm-β2M and tested for their sensitivity to lysis by clone 3C9. Some of the targets were incubated overnight at room temperature and pulsed with 100 ng Qdm before the CTL assay. b, L-Qa-1b cells were transfected with Qdm-β2M and tested as in a.

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To further ascertain that Qdm-β2M specifically associates with Qa-1b, we transfected human cell line KJ-29, which lacks β2M expression (12), with Qdm-β2M. Because this line does not express Qa-1b, we infected these cells with a rVV containing a Qa-1b insert before labeling to allow for its expression. As noted in Fig. 4, this cell line is not lysed by clone 3C9. Addition of the Qdm peptide to untransfected cells also does not result in CTL-mediated lysis because there is no β2M expression to allow for rescue of Qa-1b by this peptide. However, transfection of KJ-29 cells with Qdm-β2M renders this cell line susceptible to lysis. Transfection of β2M into line KJ-29 also does not result in target cell lysis unless the Qdm peptide is added exogenously. It is interesting to note that addition of the Qdm peptide to KJ-29 cells transfected with Qdm-β2M does not result in increased lysis, as seen with RMAS or L-Qa-1b cells (Fig. 3). This indicates that most if not all of the Qa-1b expressed on the cell surface is associated with the Qdm-β2M molecule rather than β2M, which had the Qdm portion cleaved.

FIGURE 4.

Ability of Qdm/Qa-1b-specific CTL clone 3C9 to recognize β2M KJ-29 transfectants. KJ-29 cells were infected with rVV-Qa-1b before labeling with 51Cr to allow for Qa-1b expression. KJ-29 was untransfected, or transfected with β2M or Qdm-β2M. Some targets were pulsed with 100 ng of Qdm peptide before the CTL assay.

FIGURE 4.

Ability of Qdm/Qa-1b-specific CTL clone 3C9 to recognize β2M KJ-29 transfectants. KJ-29 cells were infected with rVV-Qa-1b before labeling with 51Cr to allow for Qa-1b expression. KJ-29 was untransfected, or transfected with β2M or Qdm-β2M. Some targets were pulsed with 100 ng of Qdm peptide before the CTL assay.

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We recently generated an anti-Qa-1b-specific mAb that detects Qa-1b on the surface of lymphoblasts (17). This Ab (910) recognizes Qa-1b in the absence of peptide because it binds to Drosophila-generated sQa-1b, as demonstrated by Biacore analysis (data not shown). It does not detect Qa-1b on lymphoblasts from TAP-1 mice (17) or RMAS-Qa-1b cells (Fig. 5,a). However, incubation of RMAS-Qa-1b cells with 1 μg/ml Qdm peptide results in detection of Qa-1b with this mAb (Fig. 5,b). This staining is specific in that incubation of RMAS-Qa-1b cells up-regulates Qa-1b but not Kb or Db (Fig. 5,b). In contrast, incubation of RMAS-Qa-1b cells with a Kb- (SIINFEKL) or a Db-binding peptide (ASNENMETM) results in the up-regulation of Kb or Db (Fig. 5, c and d) respectively, but not Qa-1b. Cells transfected with Qdm-β2M also reveal expression of Qa-1b (Fig. 5,e) while RMAS cells transfected with native β2M do not (Fig. 5 f). It is interesting to note that another mAb directed against Qa-1b has been described that recognizes Qa-1b on TAP-defective cells (18, 19). This data is consistent with our previous findings that some CTL clones recognize Qa-1b on RMAS cells (20). The fact that mAb 910 does not detect Qa-1b on RMAS cells without the addition of the Qdm peptide suggests that the level of non-Qdm-associated Qa-1b is low or rapidly denatures in the absence of peptide.

FIGURE 5.

Expression of Qa-1b, Kb, or Db on RMAS-Qa-1b cells. RMAS-Qa-1b cells were untransfected, transfected with β2M or Qdm-β2M and then incubated at room temperature overnight. Cells were then incubated with peptide at 1 μg/ml for 1 h before staining with anti-Qa-1b (mAb 910), anti-Kb (AF6-88.5), or anti-Db (KH95).

FIGURE 5.

Expression of Qa-1b, Kb, or Db on RMAS-Qa-1b cells. RMAS-Qa-1b cells were untransfected, transfected with β2M or Qdm-β2M and then incubated at room temperature overnight. Cells were then incubated with peptide at 1 μg/ml for 1 h before staining with anti-Qa-1b (mAb 910), anti-Kb (AF6-88.5), or anti-Db (KH95).

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To demonstrate that the Qa-1b up-regulation was due to the association of Qa-1b with Qdm-β2M, we also stained cells transfected with the Qdm-β2M construct containing the (HIS)6 tag with an anti-HIS mAb. The data in Fig. 6 shows that these cells are stained with this Ab (Fig. 6 a). Cells transfected with β2M-(HIS)6 show weak staining. Thus, this data is consistent with the functional data showing that Qdm-Qa-1b-specific CTL recognize cells transfected with the Qdm-β2M construct.

FIGURE 6.

Expression of the HIS epitope on cells transfected with Qdm-β2M-(HIS)6. RMAS-Qa-1b cells were transfected with β2M-(HIS)6 or Qdm-β2M-(HIS)6 and tested for expression of HIS, Kb, or Db. a, Qdm-β2M-(HIS)6 transfectants. b, β2M-(HIS)6 transfectants. Anti-Kb and Db Abs same as described in Fig. 5.

FIGURE 6.

Expression of the HIS epitope on cells transfected with Qdm-β2M-(HIS)6. RMAS-Qa-1b cells were transfected with β2M-(HIS)6 or Qdm-β2M-(HIS)6 and tested for expression of HIS, Kb, or Db. a, Qdm-β2M-(HIS)6 transfectants. b, β2M-(HIS)6 transfectants. Anti-Kb and Db Abs same as described in Fig. 5.

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It has been previously reported that ∼50% of adult NK cells express CD94/NKG2AR that recognize Qdm-Qa-1b and that expression of Qdm-pulsed NK-sensitive targets renders such cells resistant to NK-mediated lysis (9, 10, 17). Therefore, we wished to determine whether Qdm-β2M-transfected cells would be protected from the activity of NK cells and compare this to the protection seen when cells express either Kb or Db. Further, it is possible that other unknown peptides bound to Qa-1b may play a role in protection from NK-mediated lysis. Thus, by the use of the Qdm-β2M construct we could assess the role of Qdm/Qa-1b alone in protecting cells from the effects of NK cells.

Because our data (17) as well as others (9, 10) demonstrates that Qa-1b cells pulsed with the Qdm peptide are protected from the lytic activity of LAK cells in vitro, we decided to test whether Qdm/Qa-1b protects targets from the in vivo activity of NK cells (15). Accordingly, RMA, RMAS, or RMAS-Qa-1b cells were labeled with 125IUdR and injected into B6 mice. Two hours later, their lungs were removed and the amount of 125I radioactivity was determined. This assay measures the activity of circulating NK cells in that susceptible targets are rapidly cleared from the lungs (21). Inoculation of RMA cells into B6 mice results in ∼25% retention of the isotope in the lungs. This result is expected because these cells express class I molecules and should be protected from NK activity. Consistent with this, isotope retention is the same in RMA-inoculated animals depleted of NK cells by injection of anti-asialo GM1 (Table I). RMAS or RMAS-Qa-1b cells, lacking surface expression of class Ia molecules as well as Qa-1b expressing Qdm should be sensitive to the lytic activity of NK cells because no class I inhibitory molecules are expressed to interact with Ly49 or CD94/NKG2AR. Indeed, lungs from mice injected with such cells contain much less radioactivity (∼10%). That this decrease in radioactivity is due to NK cells is demonstrated by the finding that pretreatment of animals with anti-asialo GM1 completely reverses this effect. RMAS or RMAS-Qa-1b cells transfected with Qdm-β2M are protected from lysis such that the radioactivity retained in the lungs at 2 h is similar to that observed in RMA cells (∼24%). No such protection is noted with RMAS or RMAS-Qa-1b cells transfected with β2M only. This suggests that expression of Qdm/Qa-1b is sufficient to protect cells from NK lung-clearing activity. However, because only ∼50% of NK cells are reported to express CD94/NKG2A, as determined by tetramer binding, we wished to compare protection mediated by Qdm/Qa-1b with peptide bound to the class Ia molecules, Kb or Db.

Table I.

Lung clearance of 125IUdR-labeled RMA and RMAS cells in B6 micea

Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 10 24.83 ± 4.98 
RMA/S – 9.72 ± 2.58 
RMA/S-Qa1bb – 11 9.75 ± 1.54 
RMA/S-Qdm-β2– 24.24 ± 5.71 
RMA/S-Qa1b-Qdm-β2– 15 23.99 ± 3.37 
RMA/S-Qa1b2mb – 11 11.78 ± 2.34 
RMA Anti-asialo GM1 23.58 ± 3.93 
RMA/S-Qa1b Anti-asialo GM1 24.55 ± 2.60 
RMA/S-Qa1b-Qdm-β2Anti-asialo GM1 23.23 ± 2.39 
RMA/S-Qa1b2Anti-asialo GM1 24.05 ± 3.80 
Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 10 24.83 ± 4.98 
RMA/S – 9.72 ± 2.58 
RMA/S-Qa1bb – 11 9.75 ± 1.54 
RMA/S-Qdm-β2– 24.24 ± 5.71 
RMA/S-Qa1b-Qdm-β2– 15 23.99 ± 3.37 
RMA/S-Qa1b2mb – 11 11.78 ± 2.34 
RMA Anti-asialo GM1 23.58 ± 3.93 
RMA/S-Qa1b Anti-asialo GM1 24.55 ± 2.60 
RMA/S-Qa1b-Qdm-β2Anti-asialo GM1 23.23 ± 2.39 
RMA/S-Qa1b2Anti-asialo GM1 24.05 ± 3.80 
a

B6 mice were untreated or treated with 20 μl anti-asialo GM1 1 day before injection of tumor cells. One million 125IUdR-labeled RMA, RMA/S, and transfectants were injected i.v., and the radioactivity in the lung was determined 2 h later.

b

, Difference between these two groups is not significant (p = 0.0798).

To address this, we pulsed RMAS-Qa-1b cells with peptide before in vivo inoculation. We demonstrated that this treatment results in up-regulation of the appropriate class I molecule (Fig. 5). Because this in vivo assay requires only 2 h, shedding and turnover of Ag should not play an important role. We show that animals injected with RMAS-Qa-1b cells pulsed with either Qdm, the Kb-binding OVA peptide (SIINFEKL) or the Db-binding influenza peptide (ASNENMETM) retain equivalent amounts of lung-associated radioactivity (24–25%), which is similar to that seen in animals inoculated with RMA cells (Table II). No protection from lung clearance is observed if the cells are unpulsed or pulsed with a control peptide, which we previously showed does not bind efficiently to Qa-1b (22). This protection by peptide-pulsed Kb is thought to be due to the interaction of these molecules with Ly49C/I inhibitory receptors (23, 24, 25, 26). It is less clear what Ly49 receptor recognizes Db. It is unlikely that it is due to the loading of the Qdm epitope from the Db leader peptide into Qa-1b because this is a TAP-dependent process (16). Although cell membrane Db does not result in binding to Ly49C or cause inhibition of NK activity in vitro, it has recently been shown that Db tetramers bind to Ly49C (23, 24, 26) and Db- donor bone marrow grafts are rejected by B6 animals (27). Because this in vivo assay is very sensitive in detecting NK-mediated lysis and its inhibition by class I ligands, this result suggests that Db can send a negative signal to NK cells through Ly49C. Future studies will address this issue. Thus, this data suggests that the expression of Qdm/Qa-1b is as effective as Kb or Db expression in protecting cells from the lytic activity of NK cells. Ly49C/I, similar to CD94/NKG2AR, are also only expressed on a subset of adult NK cells (23, 24). Thus the basis for apparent complete inhibition of NK activity at present is unknown. Because NK cells that do not bind Qdm/Qa-1b tetramers do not kill class Ihigh targets (Ref. 28 and data not shown) this suggests that tetramer cells either express low levels of receptor that are undetectable by current assays or that expression of Qdm/Qa-1b protects cells by other mechanisms.

Table II.

Lung clearance of 125IUdR-labeled RMA and RMAS cells in B6 micea

Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 25.17 ± 2.99 
RMA/S-Qa1b – 11.79 ± 1.57 
RMA/S-Qa1b OVA 25.53 ± 2.65 
RMA/S-Qa1b Influenza 24.45 ± 2.53 
RMA/S-Qa1b Qdm 23.75 ± 2.89 
RMA/S-Qa1b G13468 10.61 ± 1.77 
Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 25.17 ± 2.99 
RMA/S-Qa1b – 11.79 ± 1.57 
RMA/S-Qa1b OVA 25.53 ± 2.65 
RMA/S-Qa1b Influenza 24.45 ± 2.53 
RMA/S-Qa1b Qdm 23.75 ± 2.89 
RMA/S-Qa1b G13468 10.61 ± 1.77 
a

RMAS-Qa1b cells were untreated or incubated with a Kb binding peptide (SIINFEKL), Db binding peptide (ASNENMETM), Qa1b binding peptide (AMAPRTLLL), or control peptide G13468 (GMGGRGLGL) at a concentration of 1 μg/ml at room temperature overnight. The cells were then labeled with 125IUdR and 1 × 106 cells were inoculated i.v. The radioactivity present in the lung was determined 2 h later.

It should also be noted that in a different system that also measures in vivo NK activity (i.e., bone marrow engraftment), expression of just Kb or Db on the donor cells does not prevent their rejection in B6 mice (27). Whether this result is due to the different assay systems or the sensitivity of tumor cells vs bone marrow precursor cells in delivering signals to NK cells is unknown.

It has been reported that DBA/2 mice express relatively low levels of Qdm/Qa-1b tetramer-binding receptors (10). Therefore, it was of interest to determine whether Qdm/Qa-1b would protect RMAS-Qa-1b cells in the lung clearance assay. We pulsed RMAS-Qa-1b cells with Qdm, SIINFEKL, or ASNENMETM to determine the protective effect of Qa-1b, Kb, or Db, respectively, in DBA/2 hosts. As shown before, 125I activity is relatively high in the lungs of animals receiving RMA cells (22%; Table III). In contrast, animals receiving labeled RMAS-Qa-1b cells retained much less 125I-associated lung radioactivity (9%). RMAS-Qa-1b cells pulsed with either the Kb- or Db-binding peptide are protected from lung clearance (22–23%). However, RMAS-Qa-1b cells either pulsed with the Qdm peptide or transfected with the Qdm-β2M construct are not protected (8–10%). This demonstrates that the relative level of expression of CD94/NKG2AR plays a role in the in vivo function of NK activity and that expression of Qdm/Qa-1b might not always induce an inhibitory signal in some mouse strains.

Table III.

Lung clearance of 125IUdR-labeled RMA and RMAS cells in DBA/2 micea

Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 22.25 ± 3.68 
RMA/S-Qa1b – 8.97 ± 1.54 
RMA/S-Qa1b-Qdm-β2– 9.95 ± 2.76 
RMA/S-Qa1b OVA 23.31 ± 3.32 
RMA/S-Qa1b Influenza 22.13 ± 3.60 
RMA/S-Qa1b Qdm 7.76 ± 2.76 
Tumor CellsPretreatmentNo. of Mice% 125IUdR Retained (X ± SD)
RMA – 22.25 ± 3.68 
RMA/S-Qa1b – 8.97 ± 1.54 
RMA/S-Qa1b-Qdm-β2– 9.95 ± 2.76 
RMA/S-Qa1b OVA 23.31 ± 3.32 
RMA/S-Qa1b Influenza 22.13 ± 3.60 
RMA/S-Qa1b Qdm 7.76 ± 2.76 
a

RMA/S-Qa1b cells were untreated or incubated with OVA, influenza, or Qdm peptide in the concentration of 1 μg/ml at room temperature overnight. The cells were labeled with 125IUdR and 1 × 106 cells were inoculated. The radioactivity present in the lungs was determined 2 h later.

In summary, the Qdm peptide can be covalently linked to β2M via a flexible linker, which allows for its association on the cell surface with Qa-1b on TAP and β2M defective cells. We used this construct to demonstrate that Qdm/Qa-1b is recognized by allospecific T cells as well as inhibitory receptors on NK cells. While Qdm/Qa-1b protects RMAS and RMAS-Qa-1b cells in an in vivo lung clearance assay as effectively as peptide bound Kb or Db in B6 animals, Qdm/Qa-1b does not prevent lung clearance of RMAS-Qa-1b cells in DBA/2 hosts. Because the latter strain expresses low levels of receptor, this suggests that the level of CD94/NKG2A expression can determine whether an inhibitory signal is generated.

1

This work was supported by National Institutes of Health Grants AI45764 and AI37818.

3

Abbreviations used in this paper: Qdm, Qa-1 determinant modifier; B6, C57BL/6; β2M, β2-microglobulin; 125IUdR, 5-iodo-2′deoxyuridine-125I; rVV, recombinant vaccinia virus.

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