MHC class I molecules on adenovirus E1A-expressing tumor cells inhibit NK cell killing but not NK cell-mediated tumor rejection

John M. Routes1,2,3,4, James C. Ryan5,6, Sharon Ryan1 and Mary Nakamura5,6

1 Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA
2 Departments of Medicine and
3 Immunology, and
4 Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262, USA
5 Department of Medicine, University of California at San Francisco, San Francisco, CA 94113, USA
6 Veterans Affairs Medical Center, San Francisco, CA 94121, USA

Correspondence to: Correspondence to: J. M. Routes, National Jewish Center for Immunology and Respiratory Medicine, Department of Medicine, 1400 Jackson Street, Denver, CO 80206, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of adenovirus E1A gene products in tumor cells enhances NK cell lysis in vitro and NK-mediated rejection in vivo, despite increasing class I molecules on tumor cells. It is unclear why the increased expression of MHC class I molecules does not appear to confer resistance to killing by NK cells. One possibility is the unique capacity of E1A to sensitize cells to multiple NK cell killing mechanisms including perforin/granzyme, Fas ligand, tumor necrosis factor-{alpha} and TRAIL. To examine this issue, MCA-102-E1A tumor cells (H-2b) that express E1A and are NK sensitive were transfected with H-2Dd, the ligand for the NK inhibitory receptor, Ly49A. Expression of H-2Dd molecules by MCA-102-E1A cells protected them from lysis by a Ly49A+ NK cell clone and Ly49A+ NK cells isolated from C57BL/6 nude mice. In contrast, NK cell-mediated rejection of MCA-102-E1A tumor cells was not inhibited by the expression of H-2Dd molecules, nor was killing by polyclonal populations of NK cells isolated from C57BL/6-nude mice. H-2Dd interacts with several inhibitory Ly49 receptors that are non-clonally expressed on NK cells in C57BL/6 mice: Ly49A (20% of NK cells), Ly49G2 (54% of NK cells) and Ly49C/I (47% of NK cells). Our data indicate that while E1A sensitizes cells to NK cell killing, it does not interfere with signal transduction by inhibitory NK receptors. Therefore, a small population of NK cells that do not express Ly49A, Ly49G2 or Ly49C/I inhibitory receptors are likely responsible for the rejection of MCA-102-E1A-Dd tumor cells in vivo.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The recognition and lysis of target cells by NK cells is complex and incompletely understood. Early observations showed that the loss of expression of class I molecules on tumor cells rendered these cells sensitive to NK lysis in vitro and NK cell-mediated rejection in vivo (1,2). As a result of these observations, Kärre and Ljunggren proposed that NK cells formed a `defense system geared to detect the deleted or reduced expression of self-MHC', the missing self-hypothesis (3). This hypothesis has provided a mechanistic explanation of why NK cells kill certain class I-deficient target cells and why this killing is reversed upon transfection of the missing class I genes. Recent studies have helped to define the molecular basis for these observations. NK cells express inhibitory receptors that recognize specific MHC class I molecules (4,5). For example, in mice the NK inhibitory receptor CD94/NKG2 specifically recognizes Qa-1b, whereas the Ly49 receptor family interacts with H-2K or H-2D (4,5). Thus, target cells that express low levels of MHC class I molecules do not engage inhibitory receptors, trigger activating NK cell receptors and are sensitive to NK lysis. In contrast, target cells that express high levels of MHC class I molecules engage the inhibitory receptors on NK cells and block NK cell-mediated killing.

The adenovirus (Ad) system is a well-documented, but poorly understood, exception to the inverse relationship between the expression of class I MHC molecules on target cells and susceptibility to NK cell lysis. The expression of the Ad serotype 2 or 5 (Ad2/5) E1A gene products following either infection, transfection or viral transformation sensitizes target cells of several different species (human, mouse, rat, hamster) to lysis by NK cells (612). The ability of E1A to sensitize cells to NK cell killing is unrelated to the expression of class I molecules on target cells (9,1215). In fact, the expression of Ad5E1A gene products in E1A-transfected tumor cells or Ad2/5-transformed cells typically increases the surface expression of class I MHC molecules (12,13,15,16). Moreover, IFN-{gamma} treatment, which increases the expression of MHC class I molecules on E1A-expressing target cells, does not confer resistance to NK cell killing (14,17). Thus, in cells that express E1A, sensitivity to NK cell lysis does not appear to be inhibited by expression of high levels of MHC class I molecules.

E1A induces novel biological effects in target cells that may explain the apparent inability of MHC class I molecules to regulate NK killing. Perforin/granzyme, Fas ligand (FasL), tumor necrosis factor (TNF)-{alpha} and TRAIL all contribute to NK cell lysis of target cells (1823). In other model systems, reduction in the expression of class I molecules increases the susceptibility of cells to lysis by NK cells. However, decreases in MHC class I molecules do not change the intrinsic sensitivity of target cells to these NK killer effector mechanisms. In contrast, E1A expression sensitizes target cells to lysis by perforin/granzyme, FasL, TNF-{alpha} and TRAIL (10,24). Furthermore, perforin/granzyme, TNF-{alpha} and FasL contribute to the NK cell lysis of E1A-expressing cells (10,24,25 and unreported observations). Thus, the capacity of E1A to sensitize target cells to multiple killing mechanisms may over-ride the effects of MHC class I expression on the inhibition of NK cell activity. Alternatively, the capacity of MHC class I molecules to block the killing of E1A-expressing cells by NK cells could be masked in polyclonal populations of NK cells. Such polyclonal populations may include NK cells that are not subject to inhibition by the MHC class I molecules expressed by the target cells. Although a few studies used a clonal population of NK cells (10,12), none used clonal NK cell populations that uniformly expressed a NK cell inhibitory receptor known to interact with the particular MHC class I molecules present on the E1A-expressing target.

Recently, we demonstrated that transfection of the Ad5-E1A gene into the highly oncogenic, NK-resistant, tumor cell line, MCA-102 (H-2b), induced sensitivity to NK cell-mediated lysis. In nude mice, where rejection of tumor cells is mediated by NK cells, MCA-102-E1A tumor cells are ~100-fold less tumorigenic than parental MCA102 tumor cells (15). The levels of MHC class I molecules on the NK-sensitive, MCA-102-E1A cells were as much as 4-fold higher in comparison to the levels expressed on the parental, NK-resistant MCA-102 cells. Thus, the induction of NK cell sensitivity and the decreased tumorigenicity of MCA-102-E1A cells were not a consequence of a down-regulation of MHC class I molecules.

We adapted the MCA-102 model system to address whether expression of E1A gene products can undermine the ability of MHC class I molecules to deliver an inhibitory signal to NK cells. The killing of non-transfected and H-2Dd-transfected MCA-102-E1A cells by the NK line RNK-Ly49A was examined. RNK-Ly49A is a rat NK cell line stably transfected with the inhibitory receptor, Ly49A, which recognizes H-2Dd (26). Prior studies showed that in C57BL/6 mice, ~20% of NK cells express Ly49A (27). Therefore, cytolysis assays were also performed using Ly49A+, Ly49A and polyclonal populations of NK cells isolated from the spleens of C57BL/6 nude mice. Finally, we determined whether expression of H-2Dd inhibited the NK cell-mediated rejection of MCA-102-E1A cells by C57BL/6 nude mice, resulting in an increase in tumorigenicity of H-2Dd-expressing cells. Our results showed that expression of H-2Dd protected MCA-102-E1A cells from lysis by Ly49A+ NK cells. In contrast, H-2Dd expression did not block killing by bulk populations of NK cells nor did it inhibit the NK cell-mediated rejection of MCA-102-E1A cells by C57BL/6-nude mice.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and cell lines
The C57BL/6-derived, methylcholanthrene-induced sarcoma cell line, MCA-102, was provided by Dr Nicholas Restifo (National Institute of Health, Bethesda, MD) (28). MCA-102-E1A cells were derived by transfection of the Ad5-EIA gene and express Ad5-E1A gene products (15). To obtain H-2 Dd-expressing MCA-102-E1A cells, MCA-102-E1A were co-transfected with pSV2-Neo-Dd (29) and the hygromycin resistance vector, pLSXH, at a 10:1 molar ratio. pSV2-Dd was provided by Dr Terry Potter (National Jewish Medical and Research Center). Hygromycin-resistant MCA-102-E1A cells were screened for high-level H-2Dd expression by FACS using the anti-H-2Dd mAb, 34-5-8S (30). pLSXH-transfected, hygromycin-resistant, MCA-102-E1A (MCA-102-E1A-control) and parental MCA-102-E1A cells served as controls. RNK-Ly49 cells are a Ly49A-transfected, rat NK (RNK-16) cell line (26). The MCA-102 cell lines were maintained in DMEM supplemented with antibiotics, 15 mM glucose and 5% FCS, whereas the RNK lines were maintained in RPMI supplemented with antibiotics, 15 mM glucose and 10% FCS. Cell lines were periodically tested for contamination with Mycoplasma using the Mycotec assay (Bethesda Research Labs, Bethesda, MD) and were negative.

NK cell cytolysis assays
Six-hour, 51Cr-release, NK cytolysis assays were performed as described (17). For antibody inhibition studies, effector cells were preincubated for 15 min at room temperature with intact antibody at a concentration of 10 µg/106 effectors prior to the addition of targets. Spleen cells from athymic nude C57BL/6 mice served as the source of polyclonal NK cells. In vivo depletions of NK cells in C57BL/6 nude mice were performed using the mAb, PK136, as described (31). PK136 recognizes the NK1.1 antigen on NK cells (31). The results shown represent the mean ± SEM of at least four separate experiments. The mean percentage spontaneous release from all types of target cells was <20%.

Isolation of splenic-derived, IL-2 activated Ly49A+ and Ly49A NK cells
IL-2-activated NK cells were prepared using fresh splenocytes from C57BL/6-nude mice as previously described (32). Ly49A+ and Ly49A IL-2-activated NK cells were isolated by panning as previously described (33). Briefly, day 6 IL-2-activated NK cells were panned with the anti-Ly49A mAb, A1(34). The purity of the Ly49 NK cell population was ensured by treatment with anti-Ly49A and rabbit anti-mouse Ig (Cappel, Malvern, PA), followed by rabbit complement (Cedarlane, Westbury, NY) for 1 h at 37°C. Ly49A and Ly49A+ cell populations were then cultured overnight in complete RPMI supplemented with 1000 U/ml human IL-2 (NCI, Frederick, MD). Cells were washed extensively with HBSS with 3% FCS on day 7, replated and used for assays on day 9. This resulted in populations of NK cells that were >95% pure as assessed by their expression of NK1.1 and Ly49A by FACS.

Tumor induction studies
Congenitally athymic C57/BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Quantitative tumor induction studies were performed as previously described (35). Briefly, mice (two animals per dilution) were injected s.c. with serial log concentrations of the different MCA-102-E1A lines (MCA-102-E1A, MCA-102-E1A-control and MCA-102-E1A-Dd-CL-1) and observed weekly for tumor development for 12 weeks. Animals were sacrificed when tumors reached 20 mm mean diameter or at the end of the 12-week observation period. Tumor titrations were repeated twice. Tumor cells from animals injected with the MCA-102-E1A lines were tested for E1A expression by Western analysis. TPD50 values (log10 of the number of tumor cells required to produce tumors in 50% of the mice) were calculated by the method of Karber (36).

Measurement of MHC class I and Ly49A molecules
To measure H-2Dd expression, control and H-2Dd-transfected MCA-102-E1A cells were stained with 34-5-8 mAb (anti H2-Dd) and analyzed on an Epics C flow cytometer (17). To measure Ly49A expression, freshly isolated splenocytes were incubated with blocking 2.4.G2 mAb (anti-Fc receptor) supernatant, then stained with phycoerythrin-conjugated PK136 mAb, which recognizes the NK1.1antigen on NK cells (31), and FITC-conjugated A1 mAb, which recognizes the Ly49A receptor (34). Ly49A expression was measured on the gated population of NK1.1+ splenocytes. Ly49A expression on wild-type RNK cells and RNK-Ly49A transfectants was determined by FACS following incubation of cells with blocking 2.4.G2 mAb (anti-Fc receptor) then FITC-conjugated A1 mAb.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
High level expression of H-2Dd on MCA-102-E1A cells
Following transfection with pSV2-Dd and pLSXH, hygromycin-resistant colonies were expanded and cells were screened for H-2Dd expression by FACS. Two clones that expressed high levels of H-2Dd (MCA-102-E1A-Dd-CL1 and CL2) were chosen for subsequent analysis (Fig. 1Go). These clones expressed H-2Dd at levels nearly equivalent to P815 cells, a mastocytoma cell line that expresses high levels of H-2Dd. Hygromycin-resistant, MCA-102-E1A (MCA-102-E1A-control) served as a control cell line for these studies. Western blot analysis of cell lysates demonstrated that the amounts of E1A expressed in MCA-102-E1A-control, MCA-102-E1A-Dd-CL1 and MCA-102-E1A-Dd-CL2 were comparable (data not shown). The morphologies and in vitro doubling times of control or Dd-expressing MCA-102-E1A lines were indistinguishable from those of parental, MCA-102-E1A cells (data not shown).



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Fig. 1. Expression of H-2Dd on MCA-102-E1A cells transfected with pSV-Dd. Surface expression of H-2Dd on MCA-102-E1A-Dd-CL1 and MCA-102-E1A-Dd-CL2 was compared to the levels expressed on parental, MCA-102-E1A cells and P815 cells by FACS using the anti-H-2Dd mAb, 34-5-85. P815, a mastocytoma cell line, expresses high levels of H-2Dd, whereas parental MCA-102-E1A cells do not express H-2Dd.

 
Polyclonal NK cells, but not Ly49A+ NK cells, kill H-2Dd-expressing MCA-102-E1A cells
We first examined whether H-2Dd expression on MCA-102-E1A cells would inhibit killing by RNK-Ly49A; a rat NK cell line (RNK-1) stably transfected with the Ly49A gene. As shown in Fig. 2Go(A), H-2Dd expression on MCA-102-E1A cells did not inhibit killing by RNK cells that did not express Ly49A (Fig. 2AGo). Additionally, MCA-102-E1A-control cells were highly susceptible to killing by RNK-Ly49A cells (Fig. 2BGo). In contrast, the expression of H-2Dd on MCA-102-E1A cells inhibited killing by RNK-Ly49A cells to a level equivalent to the NK-resistant, MCA 102 cell line (Fig. 2BGo). Thus, expression of H-2Dd on MCA-102 cells induced an inhibitory signal to Ly49A-expressing RNK cells.



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Fig. 2. Lysis of E1A-expressing MCA-102-E1A cells transfected with H-2Dd by RNK and RNK-Ly49 cells. (A) NK cell lysis of MCA-102 cells, MCA-102-E1A-control and H-2Dd-expressing, MCA-102-E1A cells using RNK cells that do not express the Ly49A receptor. The E:T cell ratio in these experiments was 25:1. (B) NK cell lysis of MCA-102 cells, MCA-102-E1A-control (E1A-control) and H-2Dd-expressing, MCA-102-E1A cells (D d-CL1 and Dd-CL2) using Ly49A-transfected RNK cells.

 
Next, we determined whether expression of H-2Dd on MCA-102-E1A cells would inhibit killing by a polyclonal population of NK cells present in C57BL/6-splenocytes. As shown in Fig. 3Go, there was equivalent lysis of MCA-102-E1A-control, MCA-102-E1A-Dd-CL1 and MCA-102-E1A-Dd-CL2 cells by polyclonal NK cells. In vivo depletion of NK cells in C57BL/6 mice using the antiNK.1 antibody PK136 abolished the lytic activity present in these splenocytes (Fig. 3Go). Thus, NK cells mediated the spontaneous lytic activity present in nude mouse splenocytes.



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Fig. 3. Lysis of E1A-expressing MCA-102-E1A cells transfected with H-2Dd by a polyclonal population of NK cells. NK cell lysis of E1A-expressing MCA-102-E1A cells transfected with H-2Dd. NK cell lysis of MCA-102 cells, MCA-102-E1A-control (E1A-control) and H-2Dd-expressing, MCA-102-E1A cells (Dd-CL1 and Dd-CL2) using a polyclonal population of NK cells present in splenocytes from athymic nude C57BL/6 mice, or splenocytes depleted of NK cells in vivo by treatment with the NK cell-specific mAb, PK136 (NK depleted).

 
In C57BL/6 mice, ~20% of NK cells express Ly49A, the inhibitory receptor that recognizes H-2Dd (27,33). Therefore, it is possible that the inhibitory effect on NK cell lysis of H-2Dd may not be detected using bulk populations of NK cells. However, other explanations are also possible. For example, the level of expression of Ly49A on splenic-derived, murine NK cells may be insufficient to block NK killing. Alternatively, the negative signal delivered by the Ly49A receptor cells may be insufficient to block signals delivered by activating receptors present on murine NK cells. To address these issues, we compared Ly49A expression on RNK-Ly49A and splenic-derived NK cells by FACS. Consistent with prior observations, we found that ~22% of splenic-derived, NK1.1+ NK cells expressed Ly49A (Fig. 4Go). Furthermore, the level of Ly49A expressed on splenic-derived NK cells was greater than that expressed on RNK-Ly49A cells (Fig. 4Go). Next, we directly tested the capacity of a purified population of Ly49A+ splenic-derived NK cells to kill control and H-2Dd-expressing MCA-102-E1A cells. These experiments showed that H-2Dd expression protected MCA-102-E1A cells from lysis by L49A+, but not Ly49A splenic-derived NK cells (Fig. 5Go).



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Fig. 4. Expression of Ly49A receptor on fresh splenocytes and RNK Ly49A transfectants. (A) Following incubation with blocking 2.4.G2 mAb (anti-Fc receptor) supernatant, splenocytes were stained with phyccoerythrin-conjugated PK136 mAb, which recognizes the NK1.1antigen on NK cells (31), and FITC-conjugated A1 mAb, which recognizes and the Ly49A receptor (34). Ly49 expression was measured on the gated population of NK1.1+ cells. (B) Ly49A expression on wild-type RNK-16 cells (dotted line) and RNK Ly49A transfectants (solid histogram).

 


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Fig. 5. Lysis of E1A-expressing MCA-102-E1A cells transfected with H-2Dd by splenic-derived Ly49A+ or Ly49A NK cells. Ly49A+ and Ly49A IL-2-activated NK cells were tested in 4-h cytotoxicity assays against MCA102-E1A cells (A and B), MCA-102-E1A-Dd-CL1 (C and D) or MCA-102-E1A-Dd-CL2 (E and F). Lysis by Ly49A+ NK cells is shown on the left (A, C and E) and lysis by Ly49A NK cells is shown on the right (B, D and F). Assays were performed in the presence of isotype-matched control antibody (anti-gp42, 3G7, closed circles or closed inverted triangles) or anti-Ly49A (A1, open squares or open triangles).

 
In summary, these data indicate that expression of H2Dd on target cells inhibited the killing by RNK cells and splenic-derived murine NK cells expressing L49A. However, the inhibitory effect of H-2Dd expression on Ly49A+ NK cells was not detected in cytolysis assays using bulk populations of splenic-derived NK cells.

Control and H-2Dd-expressing MCA-102-E1A cells have equivalent tumorigenicities in nude mice
Previous studies showed that the reduced tumorigenicity of MCA-102-E1A, compared to MCA-102 cells in C57BL/6-nude mice was dependent on NK cells. MCA-102-E1A cells were ~100-fold less tumorigenic than parental MCA-102 cells in nude mice, which lack T cells but express NK cells. In contrast, MCA-102-E1A and MCA-102 cells were equivalently tumorigenic in CD3{varepsilon}-transgenic mice, which lack both NK cells and T cells (15).

Next, we determined whether H-2Dd expression inhibited NK cell-mediated rejection of MCA-E1A cells in C57BL/6-nude mice. To test this possibility, we compared the tumorigenicities of MCA-102-E1A cells, MCA-102-E1A-control and MCA-102-E1A-Dd-CL1 cells in C57BL/6-nude mice. MCA-102-E1A cells are the parental cell lines for both MCA-102- E1A-control and MCA-102-E1A-Dd-CL1 cells. MCA-102-E1A-Dd-CL1 and MCA-102-E1A-control cells both express the hygromycin resistance vector, pLSXH (see Methods). Nude mice were injected s.c. with serial log concentrations of MCA-102-E1A, MCA-102-E1A-control and MCA-102-E1A-Dd-CL1 cells, and observed weekly for tumor development. TPD50 values (log10 of the number of tumor cells required to produce tumors in 50% of the mice) were calculated by the method of Karber (36). The TPD50 values of parental, MCA102-E1A cells (TPD50 = 4.0), MCA-E1A-control (TPD50 = 3.3) and MCA-102-E1A-Dd-CL1 (TPD50 = 3.7) were not significantly different (Fig. 4Go). These values were similar to the TPD50 values we previously reported on parental, MCA-102-E1A cells in nude mice (15). Therefore, the NK cell-mediated rejection of MCA-102-E1A tumor cells was not impaired by expression of H-2Dd.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Results from this study provide insight into prior observations on the relationship between expression of MHC class I molecules, tumorigenicity and NK sensitivity of Ad-transformed and E1A-transfected tumor cells. Through the expression of the E1A and E1B oncogenes, Ad are able transform mammalian cells from many species, including man. The expression of the E1A oncogene derived from group C Ad (Ad serotypes 2 and 5) also sensitizes cells of several different species (human, mouse, rat and hamster) to NK cell lysis. Studies in rodent models indicate that low oncogenicity of group C Ad-transformed cells is, in part, due to the rejection of these cells by NK cells (9,13,15). Similarly, the stable transfection of the Ad5-E1A gene into NK-resistant tumors reduces tumorigenicity as a result of enhanced NK cell-mediated rejection in vivo (15,35). However, neither the NK sensitivity nor tumorigenicity of E1A-expressing tumor cells correlated with the level of class I molecules expressed on these cells (6,9,13,15,37).

Our results showed that H-2Dd expresssion on MCA-102-E1A cells inhibited killing of target cells by Ly49A+ RNK cells (Fig. 2BGo) and Ly49A+ splenic-derived NK cells (Fig. 5Go). In contrast, H-2Dd expression did not measurably influence the killing of MCA-102-E1A cells by a polyclonal population of splenic-derived NK cells (Fig. 3Go). Based on these results, the previously reported inability of increased class I molecule expression to block NK killing on E1A-expressing cells was likely due to the masking of this effect when polyclonal populations of NK cells were used. However, consistent with the previous studies, we found that the ability of NK cells to reject E1A-expressing tumor cells in vivo best correlated with in vitro cytolysis assays using polyclonal populations of NK cells present in the tumor challenge recipient (Figs 3 and 6GoGo).



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Fig. 6. Effect of H-2Dd expression on the tumorigenicity of MCA-102-E1A cells in nude mice. MCA-102-E1A-control cells are hygromycin-resistant MCA-102-E1A cells. MCA-102-E1A cells are the parental cell lines for MCA-102-E1A-Dd-CL1 and MCA-102-E1A-control cells. Nude mice were injected s.c. with serial log concentrations of MCA-102-E1A, MCA-102-E1A-control and MCA-102-E1A-Dd-CL1 cells and observed weekly for tumor development for 3 months. The TPD50 is the log10 of the number of tumor cells required to produce tumors in 50% of the mice.

 
It is unclear how the expression of Ad2/5-E1A gene products results in the enhanced recognition and lysis by NK cells. However, the sensitivity of target cells to NK cell lysis is likely mediated by a balance of activating and inhibitory signals mediated by NK receptors. Thus, the induction of target cell susceptibility by E1A may be due to either an enhancement of the positive signal or suppression of an inhibitory signal transduced by NK cell receptors. Our data indicates that inhibitory signals are not altered by E1A expression. Therefore, we speculate that E1A expression increases target cell killing by enhancing positive signals through activating NK cell receptors.

Apart from Ly49A, H-2Dd interacts with other Ly49 receptors that are non-clonally expressed on NK cells in C57BL/6 mice. These receptors include the inhibitory receptors Ly49G2 (expressed on 54% of NK cells) and Ly49C/I (expressed on 47% of NK cells), as well as the activating NK cell receptor Ly49D (expressed on 50% of NK cells) (27,38). Our data indicated that NK cells that co-expressed inhibitory and activating NK cell receptors could not lyse Dd-expressing MCA-102-E1A cells. Thus, it is likely that in C57BL/6 nude mice a relatively small population of NK cells that do not express inhibitory receptors are sufficient to lyse large numbers of H-2Dd-expressing, MCA-102-E1A cells. Furthermore, because H-2Dd expression did not measurably affect the NK cell-dependent rejection of MCA-102-E1A cells, small numbers of uninhibited NK cells are also sufficient for the rejection of E1A-expressing tumor cells in vivo (Fig. 6Go). These results illustrate the biological importance of having NK cells that express overlapping sets of inhibitory and excitatory receptors.

In summary, H-2Dd expression on E1A-expressing MCA-102 cells inhibited killing by NK cells that expressed the Ly49A receptor. However, NK cell-dependent tumor rejection of MCA-102-E1A cells was not altered by H-2Dd expression. Thus, in an immunocompetent animal, polyclonal populations of NK cells allow for heightened tumor immunosurveillance.


    Acknowledgments
 
This work was supported by Public Health Services grants, RO1-CA76491 (J. R.), K11-ARO1927 (M. C. N.), RO1-A144126 (J. C. R.) and the Veterans Administration (M. C. N. and J. C. R.). We thank Dr Terry Potter for the plasmid pSvDd and the mAb 34-5-8, and Dr Nicholas Restifo for the MCA-102 line. We also thank Dr Terry Potter and Dr Tanya Miura for critical review of this manuscript and Gabriele Cheatham for secretarial assistance.


    Abbreviations
 
Ad adenovirus
FasL Fas ligand
TNF tumor necrosis factor

    Notes
 
Transmitting editor: W. M. Yokoyama

Received 5 February 2001, accepted 11 July 2001.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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