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The Journal of Immunology, 2000, 165: 6142-6147.
Copyright © 2000 by The American Association of Immunologists

Comparative Ability of Qdm/Qa-1b, Kb, and Db to Protect Class Ilow Cells from NK-Mediated Lysis In Vivo1

Song Hui Jia, Zoran Kurepa, Ailin Bai and James Forman2

Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results and Discussion
 References
 
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 {beta}2-microglobulin ({beta}2M) by a flexible linker (Qa-1 determinant modifier (Qdm)-{beta}2M). When this construct is expressed in TAP-2- or {beta}2M- cells, it allows for the expression of a Qdm-{beta}2M 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-{beta}2M cells into C57BL/6 mice and measured their NK cell-mediated clearance from the lungs at 2 h. RMAS cells transfected with Qdm-{beta}2M were resistant to lung clearance, similar to RMA cells or RMAS cells in anti-asialo-GM1-treated mice, while untransfected or {beta}2M-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.


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results and Discussion
 References
 
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 {beta}2M 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 {beta}2M. 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.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results and Discussion
 References
 
Animals

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

Cell lines

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 {beta}2M expression. Other tumor lines have been previously described (13). CTL clone 3C9 recognizes the Qdm peptide (AMAPRTLLL) associated with Qa-1b (7).

Construction of eukaryotic expression plasmids

Mouse {beta}2M cDNA from BALB/c mice was used as a template to generate a {beta}2M molecule containing a flexible linker attached to the Qdm nonamer peptide (Qdm-{beta}2M). Our approach is a modification of that previously described by Uger and Barber (14). Qdm-{beta}2M was generated by overlapping PCR (Fig. 1Go). To generate the 5' end of the construct (template 1) containing the {beta}2M 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 {beta}2M 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 {beta}2M protein (template 2), wild-type {beta}2M was amplified using primer 7, 5'-TCTGGAGGAGGATCTATCAAAACCCCTCAAATTC-3', which hybridizes to {beta}2M 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-{beta}2M product was cloned into the pcDNA 3.1(+) vector containing the neomycin resistance gene.



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  		FIGURE 1. Generation of the Qdm-linker-{beta}2M and Qdm-linker-{beta}2M-HIS gene constructs. See text for details.

 
To insert a (HIS)6 tag at the C-terminal end of the construct, we amplified Qdm-{beta}2M 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 {beta}2M 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.

Transfections

Three micrograms of {beta}2M or the Qdm-{beta}2M plasmid were transfected into 106 RMAS, 2 x 105 Ltk, and 2 x 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 x 106) and L-Qa-1b (2 x 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.

Western blotting

Cells (5 x 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).

CTL assays

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.

Flow cytometry

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).

Lung clearance assay

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 x 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.


   Results and Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results and Discussion
References
 
Generation of Qdm-{beta}2M constructs

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



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  		FIGURE 2. Western blot analysis of anti-HIS-binding proteins. RMAS or RMAS-Qa-1b cells were transfected with {beta}2M-(HIS)6 or Qdm-{beta}2M-(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, {beta}2M-(HIS)6; lanes 3 and 6, Qdm-{beta}2M-(HIS)6.

 
Association of Qdm-{beta}2M with Qa-1b

The above data show that a Qdm-{beta}2M 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 {beta}2M (control) construct allow for this clone to recognize such target cells (Fig. 3Goa). However, transfection of RMAS-Qa-1b cells with Qdm-{beta}2M 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-{beta}2M 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 {beta}2M. A similar result is observed with L-Qa-1b cells that do not express the Qdm epitope. Transfection with Qdm-{beta}2M allows for recognition by Qdm-Qa-1b-specific CTL (Fig. 3Gob).



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  		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 {beta}2M or Qdm-{beta}2M 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-{beta}2M and tested as in a.

 
To further ascertain that Qdm-{beta}2M specifically associates with Qa-1b, we transfected human cell line KJ-29, which lacks {beta}2M expression (12), with Qdm-{beta}2M. 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. 4Go, 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 {beta}2M expression to allow for rescue of Qa-1b by this peptide. However, transfection of KJ-29 cells with Qdm-{beta}2M renders this cell line susceptible to lysis. Transfection of {beta}2M 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-{beta}2M does not result in increased lysis, as seen with RMAS or L-Qa-1b cells (Fig. 3Go). This indicates that most if not all of the Qa-1b expressed on the cell surface is associated with the Qdm-{beta}2M molecule rather than {beta}2M, which had the Qdm portion cleaved.



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  		FIGURE 4. Ability of Qdm/Qa-1b-specific CTL clone 3C9 to recognize {beta}2M- 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 {beta}2M or Qdm-{beta}2M. Some targets were pulsed with 100 ng of Qdm peptide before the CTL assay.

 
Cell membrane expression of Qa-1b on transfectants

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. 5Goa). However, incubation of RMAS-Qa-1b cells with 1 µg/ml Qdm peptide results in detection of Qa-1b with this mAb (Fig. 5Gob). This staining is specific in that incubation of RMAS-Qa-1b cells up-regulates Qa-1b but not Kb or Db (Fig. 5Gob). 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. 5Go, c and d) respectively, but not Qa-1b. Cells transfected with Qdm-{beta}2M also reveal expression of Qa-1b (Fig. 5Goe) while RMAS cells transfected with native {beta}2M do not (Fig. 5Gof). 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.



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  		FIGURE 5. Expression of Qa-1b, Kb, or Db on RMAS-Qa-1b cells. RMAS-Qa-1b cells were untransfected, transfected with {beta}2M or Qdm-{beta}2M 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).

 
To demonstrate that the Qa-1b up-regulation was due to the association of Qa-1b with Qdm-{beta}2M, we also stained cells transfected with the Qdm-{beta}2M construct containing the (HIS)6 tag with an anti-HIS mAb. The data in Fig. 6Go shows that these cells are stained with this Ab (Fig. 6Goa). Cells transfected with {beta}2M-(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-{beta}2M construct.



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  		FIGURE 6. Expression of the HIS epitope on cells transfected with Qdm-{beta}2M-(HIS)6. RMAS-Qa-1b cells were transfected with {beta}2M-(HIS)6 or Qdm-{beta}2M-(HIS)6 and tested for expression of HIS, Kb, or Db. a, Qdm-{beta}2M-(HIS)6 transfectants. b, {beta}2M-(HIS)6 transfectants. Anti-Kb and Db Abs same as described in Fig. 5Go.

 
Functional significance of Qdm-Qa-1b surface expression

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-{beta}2M-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-{beta}2M 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 IGo). 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-{beta}2M 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 {beta}2M 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.


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  		Table I. Lung clearance of 125IUdR-labeled RMA and RMAS cells in B6 mice1

 
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. 5Go). 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 IIGo). 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.


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  		Table II. Lung clearance of 125IUdR-labeled RMA and RMAS cells in B6 mice1

 
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.

Ability of Qdm to protect cells from lysis in animals expressing low levels of Qdm/Qa-1bR.

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 IIIGo). 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-{beta}2M 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.


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  		Table III. Lung clearance of 125IUdR-labeled RMA and RMAS cells in DBA/2 mice1

 
In summary, the Qdm peptide can be covalently linked to {beta}2M via a flexible linker, which allows for its association on the cell surface with Qa-1b on TAP and {beta}2M 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.


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

2 Address correspondence and reprint requests to Dr. James Forman, Center for Immunology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines, Dallas, TX 75390-9093. Back

3 Abbreviations used in this paper: Qdm, Qa-1 determinant modifier; B6, C57BL/6; {beta}2M, {beta}2-microglobulin; 125IUdR, 5-iodo-2'deoxyuridine-125I; rVV, recombinant vaccinia virus. Back

Received for publication June 21, 2000. Accepted for publication August 30, 2000.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results and Discussion
 References
 

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