International Immunology

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International Immunology, Vol. 12, No. 6, 843-850, June 2000
© 2000 Japanese Society for Immunology

Self-MHC class Ia (RT1-Aⁿ) protects cells co-expressing the activatory allogeneic MHC class Ib molecule (RT1-E^u) from NK lysis

Eva Bäckman-Petersson¹, Geoffrey W. Butcher² and Gunnar Hedlund^1,3

¹ Department of Tumor Immunology, Immunobiology, BMC, Lund University, Sölvegatan 21, 223 62 Lund, Sweden
¹ Molecular Immunology Program, The Babraham Institute, Cambridge CB2 4AT, UK
¹ Active Biotech Research, 223 63 Lund, Sweden

Correspondence to: E. Bäckman-Petersson

	Abstract

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We have previously shown activation of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E^u. In this study we investigated whether a self-MHC class I molecule could protect the allogeneic targets from being recognized and killed by the alloreactive NK (allo NK) cells. NK cells from BN (RT1 n) rats, primed in vivo by immunization with RT1^u-expressing cells, manifested cytolytic activity against RT1^u- as well as RT1^u/lv1-expressing targets, but not against RT1^u/n-expressing targets. The absence of cytolytic activity against semiallogeneic targets, i.e. targets expressing self-allotypes, was also valid for allo NK cells from alloimmunized F344 (RT1 ^lv1) rats. To analyze the ability of a distinct MHC class I molecule to protect target cells from NK lysis, Rat2 cells transfected with the activating allogeneic MHC class Ib, RT1-E^u molecule were also transfected with the self-MHC class Ia, RT1-A1ⁿ molecule. The allo NK cells from BN rats immunized with RT1^u-expressing cells were cytolytic against Rat2 transfected with the RT1-E^u molecule. However, the allo NK cells manifested no cytolytic activity against double-transfected Rat2 cells, expressing the RT1-E^u as well as the RT1-A1ⁿ molecule. We conclude that expression of a self-MHC class Ia (RT1-A) molecule protects targets from allo NK killing. Furthermore, the NK inhibition via recognition of the self-MHC class Ia molecule dominates over the activation via recognition of the allogeneic MHC class Ib molecule, RT1-E.

Keywords: activation, inhibition, MHC class I, NK cells

	Introduction

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NK cell-mediated cytolysis was first shown to correlate with decreased expression of MHC class I molecules, such as in tumors and virus infections. The missing-self recognition model first proposed by Kärre et al. stated that, in contrast to cytotoxic T lymphocytes (CTL), which require recognition of MHC to activate killing, NK cells were inhibited when recognizing MHC class I (1 and reviewed in 2). The concept of NK killing of target cells lacking MHC class I has later been extended to include killing of target cells failing to express MHC class I of a specific allotype (3,4). The inhibition of cytolysis upon specific MHC class I recognition is mediated through NK inhibitory receptors. Primarily three groups of NK inhibitory receptors have been characterized: the lectin-like homodimeric Ly49 receptor family (reviewed in 5–7), the lectin-like heterodimeric CD94/NKG2 receptors (8–10 and reviewed in 7) and the killer inhibitory receptors of the Ig superfamily (11,12 and reviewed in 7,13). The cytoplasmic domain of the inhibitory receptors contains immunoreceptor tyrosine-based inhibitory motifs to which the SHP-1 tyrosine phosphatases are recruited (reviewed in 14,15). Recently, molecules homologous to the inhibitory receptors have been shown to activate NK cells (16–19). These triggering receptors are coupled to dimers of the DAP-12 protein, which contain a tyrosine-based activation motif similar to the chain of the TCR–CD3 complex (20,21). In the rat, gene homologues of human CD94 (22) and NKG2 (23) exist, and an inhibitory receptor homologous to mouse Ly49 has been characterized (24).

We have previously shown that BN (RT1ⁿ) rats manifested alloreactive NK cells (CD2⁺CD3^– TCR^– NKR-P1⁺) after i.p. immunization with allogeneic WF (RT1^u) cells (25). The NK population induced in this way, which we term allo NK cells, appears to have been either selectively recruited or expanded in an alloantigen-specific fashion. By genetic studies, we and others have shown that rat NK cells are activated by structures encoded within the non-classical MHC class Ib, RT1-E/C region (26,27). Furthermore, the allogeneic RT1-E^u molecule was identified as a target structure for NK cells (27). In addition to the experimental evidence for NK activation via recognition of MHC class Ib molecules, we have shown that inhibition of allo NK cytolytic activity correlated with expression of self-MHC (25). Inhibition due to recognition of self-products has also been suggested when analyzing NK killing of target cells from intra-MHC recombinant rat strains (28 and reviewed in 29).

In this study we have extended our analysis of the role of self-MHC in regulating the activation of the in vivo primed allo NK cells. Since the NK inhibitory function has been associated with the classical RT1-A region (28), we have analyzed the RT1-A1ⁿ molecule (30), which has the expected properties of a principal MHC class Ia molecule (reviewed in 31), for its ability to inhibit allo NK cells. An additional MHC class I RT1-A molecule of the n haplotype, termed RT1-A2ⁿ, has also been identified, but its functional status is, as yet, much less clear (30). Both semiallogeneic cells co-expressing a self-RT1 haplotype together with an allogeneic RT1 haplotype, and Rat2 fibroblast cells transfected with RT1-E^u and/or RT1-A1ⁿ were tested for sensitivity to allo NK killing. Novel results are presented regarding the role of distinct RT1 molecules in the activation and inhibition of this in vivo selected allo NK population.

In addition, in order to investigate whether allo NK cells could be activated in an additional rat strain, and thus be regarded as a general phenomenon, we immunized F344 (RT1^lv1) rats with RT1^u-expressing cells and analyzed the selective cytolytic activity of distinct lymphocyte subsets.

	Methods

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Animals and cell lines
Rats of the BN (RT1ⁿ), WF (RT1^u) and F344 (RT1^lv1) strains were bred at the Department of Tumor Immunology, Lund University. The designation F₁ (RT1^u/n) denotes the first generation offspring of WF (female)xBN (male) cross-matings. The designation F₁ (RT1^u/lv1) denotes the first generation offspring of WF (female)xF344 (male).

The Rat2 cell line, derived from a subclone of the F344 (RT1^lv1) rat fibroblast Rat1 (32), was obtained from ATCC (Manassas, VA). The Rat2 cells were cultured in R10 medium composed of RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10 mM HEPES (Sigma, St Louis, MO), 2 mM L-glutamine (Gibco), 1 mM pyruvate and 10% FCS (Flow, Irvine, UK), at 37°C in a humidified atmosphere of 5% CO₂.

Immunization procedure and peritoneal wash
For immunization, four doses of 10⁷ allogeneic spleen cells were inoculated i.p. at intervals of 1 week into responder rats. Peritoneal washes were performed 3–4 days after the last inoculation. The skin was cut away from a ventral area of the abdomen and 30 ml PBS was injected into the peritoneal cavity. After inoculation, the abdomen was massaged gently, the needle was reintroduced and the buffer containing peritoneal cells was aspirated.

Cytofluorometric analysis and cell sorting
The cells were stained with FITC-, phycoerythrin- and biotin-conjugated antibodies directed against cell-surface molecules. A mouse mAb directed against rat CD3 (G4.18) and streptavidin–phycoerythrin were purchased from PharMingen (San Diego, CA). The mouse 3.2.3 mAb directed against NKR-P1 and the NR3/31 rat mAb directed against the RT1-A^u molecule were purchased from Serotec (Oxford, UK). The goat anti-rat F(ab')₂ reagent was obtained from Jackson ImmunoResearch (Baltimore, MD). Professor Thomas Gill (Pittsburgh, PA) kindly provided the mAb 70. The mAb DN2/4 against RT1-A1ⁿ has been described previously (30). Cell staining was performed in PBS with 1% BSA (PBS/BSA) on ice. Cells were washed twice in PBS/BSA after each incubation step and kept on ice until analyzed.

Peritoneal cells were sorted on a FACS Vantage flow cytometer (Becton Dickinson, Mountain View, CA) by the distinct expression of NKR-P1 and/or CD3. Purity of sorted cells was always >95%. Cell phenotype was analyzed with a FACScan flow cytometer (Becton Dickinson). Cell phenotyping and sorting were performed with logarithmic amplification and optimal electronic compensation. Lymphocytes were gated by forward and side scatter characteristics.

RT1-A^u or RT1-E^u transfection of Rat2 cells
The cDNAs encoding the RT1-A^u and RT1-A1ⁿ MHC class I molecules were isolated from the PVG-RT1^u and the PVG-RT1ⁿ rat strains respectively using a PCR-based method (30,33). The RT1-A^u or the RT1-A1ⁿ cDNA was introduced into the retroviral vector pLXLJ, which carries a neo gene and the SV40 and pBR origins of replication (34). The pLAuLJ and pLAnLJ vectors were used for transfection into psi2 cells, which produce ecotropic virions. The psi2 cells were grown in DMEM (Gibco) supplemented with 10% newborn calf serum (Sigma) and 1.0 mg/ml Geniticin (G418; Gibco), and analyzed for RT1-A^u and RT1-Aⁿ expression (data not shown) before using the virions produced to infect the Rat2 cells. Rat2 cells (0.2x10⁶) in R10 were seeded in a 25 cm² plastic bottle. One day after seeding the medium was removed, and the cells were incubated with 2 ml virus supernatant diluted 1:5 and supplemented with 4 µg/ml hexadimetrine bromide (polybren; Sigma) for 5 h. Thereafter 3 ml R10 medium was added and the cells left overnight. One day after virus infection new R10 medium was added and after 2 days the cells were supplemented with 0.5 mg/ml Geneticin (Gibco).

The cDNA encoding the RT1-E^u MHC class I heavy chain cloned into the expression pRep10 vector (Invitrogen, San Diego, CA) was kindly provided by Professor Thomas Gill (35). A method using lipofectamine was adopted to transfect the Rat2 cells or Rat2 cells infected with the RT1-A1ⁿ cDNA. Rat2 cells or Rat2-RT1-A1ⁿ cells (0.2x10⁶) in R10 were seeded in a 25 cm² plastic bottle. The medium was removed the day after and 1.6 ml optimem (Gibco) was added. Solution A [10 µl lipofectamine reagent (Gibco), 4 µl of 10 mg/ml holotransferrin (Sigma) and 186 µl optimem] was mixed with solution B (2 µl of 1 µg/ml plasmid DNA and 198 µl optimem) and incubated for 30 min at room temperature. The Rat2 cells or Rat2-RT1-A1ⁿ cells were incubated with the RT1-E^u plasmid solution for 3 h. Thereafter 3 ml R10 was added and the cells left overnight. One day after transfection new R10 medium was added and after 2 days the cells were supplemented with 150 µg/ml hygromycin (Boehringer Mannheim, Mannheim, Germany).

Flow cytometry was used to screen the transfected Rat2 cells for surface expression of MHC class I molecules.

Assay for cell-mediated cytotoxicity
Cell-mediated cytolysis was measured with a ⁵¹Cr-release assay (36). Peritoneal cells from naive rats of different strains, non-transfected as well as transfected Rat2 cells were used as targets. ⁵¹Cr -labeled target cells (3.0x10³) were incubated for 5 h at 37°C in a humidified atmosphere of 5% CO₂ in air with 10–300x10³ effector cells in 100 µl R10 medium in plates with 96 cone-shaped wells (Nunc, Roskilde, Denmark). After incubation, 25 µl supernatant was collected and counted in a Wallac 1450 MicroBeta counter as a measure of released ⁵¹Cr. Spontaneous release was estimated by addition of R10 medium to ⁵¹Cr -labeled target cells and maximum releasable ⁵¹Cr was estimated by the addition of 1% SDS. Specific cytotoxicity was calculated in percent thus: [( mean c.p.m. released with test cells – mean c.p.m. spontaneous release)/(mean c.p.m. maximum release – mean c.p.m. spontaneous release)]x100. Mean c.p.m. was calculated from triplicate wells for effector cells and from six parallel wells each for spontaneous and maximum release. For antibody inhibition studies, the target cells were preincubated with the mAb DN2/4 (IgG2b, anti-RT1-A1ⁿ) at a concentration of 40 µg/ml (20 µg/10⁶ cells) for 30 min at room temperature prior to the addition of effector cells. To decrease the probability of antibody dependent cell-mediated cytotoxicity, the effector cells were incubated with 2% of heat-aggregated IgG (5 mg/ml) before the addition of target cells.

	Results

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Activation of allo NK cells in BN as well as F344 rats immunized with allogeneic cells
To confirm previous results showing activation of allo NK cells in BN rats (25) and to elucidate whether this response could also be shown in additional rat strains, BN as well as F344 rats were immunized i.p. with WF cells. The peritoneal cells were sorted into CD3⁺ T cells or CD3^– NKR-P1⁺ NK cells and analyzed for cytolytic activity without any further in vitro re-stimulation. Neither CD3⁺ nor CD3^– NKR-P1⁺ cells from naive BN or F344 rats were cytolytic against the target cells tested (data not shown). CD3^– NKR-P1⁺ cells, from BN anti-WF rats, were cytolytic against allogeneic WF (RT1^u) (Fig. 1C), but not against syngeneic BN or third-party allogeneic F344 target cells (Fig. 1A and B). The same result, showing cytolytic activity against allogeneic WF (Fig. 2C), but not against syngeneic or third-party allogeneic target cells (Fig. 2A and B), was also demonstrated with CD3^– NKR-P1⁺ cells from F344 anti-WF rats. The significant increase in proportion of peritoneal CD3^– NKR-P1⁺ cells in BN rats immunized with WF cells compared to naive rats (25) was also shown for F344 anti-WF rats (40% increase in the proportion of CD3^– NKR-P1⁺ compared to naive F344 rats). The activation of allo NK cells after alloimmunization could thus be shown in the F344 as well as in the BN responder rat strain. In contrast to BN anti-WF rats, which manifested no allocytolytic CD3⁺ cells (Fig. 1), CD3⁺ cells from F344 anti-WF rats manifested selective allocytolytic activity (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Cytolytic activity of peritoneal cells from BN (RT1n) rats immunized i.p. with WF (RT1u) spleen cells, BN anti-WF. The peritoneal cells were sorted into CD3+ and CD3– NKR-P1+ lymphocytes. Peritoneal cells from naive BN, RT1n (A), F344, RT1lvl (B), WF, RT1u (C), WF/BN, RT1u/n (D) and WF/F344, RT1u/lv1 (E) rat strains were used as target cells. The mean ± SD of three experiments is shown.

 

View larger version (18K): [in this window] [in a new window]   Fig. 2. Cytolytic activity of peritoneal cells from F344 (RT1lv1) rats immunized i.p. with WF (RT1u) spleen cells, F344 anti-WF. The peritoneal cells were sorted into CD3+ and CD3– NKR-P1+ lymphocytes. Peritoneal cells from naive BN, RT1n (A), F344, RT1lvl (B), WF, RT1u (C), WF/BN, RT1u/n (D) and WF/F344, RT1u/lv1 (E) rat strains were used as target cells. The mean ± SD of three experiments is shown.

 
The allo NK cells manifested no cytolytic activity against semisyngeneic target cells expressing self-MHC class I
To confirm previous results, showing absence of allo NK cytolytic activity against semiallogeneic target cells (25), and to elucidate whether this response could also be shown for allo NK cells of F344 anti-WF rats, cells from WF/BN and WF/F344 F1 progeny strains were tested for sensitivity to killing by in vivo activated NK cells. CD3^– NKR-P1⁺ cells from BN (RT1ⁿ) anti-WF (RT1^u) rats were cytolytic against WF/F344 (RT1^u/lv1), but not against WF/BN (RT1^u/n) targets (Fig. 1D and E). Furthermore, CD3^– NKR-P1⁺ cells from F344 (RT1^lv1) anti-WF (RT1^u) rats were cytolytic against WF/BN (RT1^u/n), but no significant cytolysis against WF/F344 (RT1^u/lv1) targets was recorded (Fig. 2D and E). In contrast to the CD3^– NKR-P1⁺ cells, CD3⁺ cells from F344 (RT1^lv1) anti-WF (RT1^u) rats were cytolytic against WF/BN (RT1^u/n) as well as WF/F344 (RT1^u/lv1) targets. The results show that the CD3^– NKR-P1⁺ NK cells lysed cells from F₁ progeny of the immunizing strain and a strain of another MHC haplotype. However, if the target cells were derived from F₁ progeny of the immunizing and the responding (self) strain no lysis was recorded. Previous studies showed that target cells from F₁ (WF/BN)xWF backcross progeny that lacked expression of RT1ⁿ (self-MHC class I) were susceptible to lysis, whereas those from RT1ⁿ-positive littermates were protected from lysis (25). These results indicated that expression of self-MHC molecules conferred protection from lysis

The self-MHC class I molecule RT1-A inhibits NK allocytolytic activity
Since our results showed that the allo NK cells were inhibited by recognition of target products expressed by semiallogeneic cells and this inhibition correlated with self-MHC expression (25), we analyzed the role of distinct self-MHC class I molecules in the regulation of allo NK cytolysis.

Rat2 transfectants were prepared using cDNAs of the class Ia allele, RT1-A^u, and one class Ib allele, RT1-E^u, present in the immunizing strain (WF) in our system; similarly a Rat2 transfectant of RT1-A1ⁿ, a class Ia allele of the responding BN strain, was generated. Finally, a double-transfectant expressing both RT1-A1ⁿ and RT1-E^u was generated. The transfected Rat2 cells expressed the expected surface molecules as analyzed by flow cytometry (Fig. 3). The double RT1-E^u/A1ⁿ-transfected Rat2 cells expressed the same levels of RT1-E^u and RT1-A1ⁿ molecules as the single transfected cells. [Note the mAb 70 against the RT1-E^u molecule is cross-reactive with the RT1-A^u molecule (37).]

View larger version (22K): [in this window] [in a new window]   Fig. 3. Flow cytometric analysis of transfected Rat2 cells. The Rat2 cells were transfected with the RT1-A1n, RT1-Eu, RT1-Eu/A1n and RT1-Au cDNA, and then assayed for their binding with specific mAb. The histograms represents the binding of mAb DN2/4, which is specific for the RT1-An molecule; mAb 70, which is specific for the RT1-Eu molecule, but cross-reacts with the RT1-Au molecule; and mAb NR3/31, which is specific for the RT1-Au molecule. No non-specific binding of streptavidin–phycoerythrin (which was used as a second step on biotinylated mAb 70) or goat anti-rat F(ab')2 labeled with phycoerythrin (which was used as second step on DN2/4 and NR3/31) was recorded on the transfected cells. Non-transfected Rat2 cells stained with the different mAb were used as negative controls.

 
BN rats immunized i.p. with WF (RT1^u) cells were analyzed for cytolytic activity against the immunizing cells, Rat2 cells and the various Rat2 transfectants. The CD3⁺ T cell population manifested no cytolytic activity against any of the targets tested (Fig. 4). As previously shown, CD3^– NKR-P1⁺ cells from BN anti-WF rats manifested no cytolytic activity against non-transfected Rat2 cells. However, both allogeneic WF targets and Rat2 cells transfected with RT1-E^u were killed efficiently (Fig. 4A and D), revealing the allogeneic MHC class Ib molecule to be a target for activation of NK cytolysis. Although the RT1-A^u molecule was shown to be a target molecule on transfected Rat2 cells for alloreactive CTL (27), allo NK cells did not lyse Rat2 cells expressing the allogeneic RT1-A^u (Fig. 4C). Furthermore, Rat2 cells transfected with RT1-A1ⁿ were resistant to lysis by the CD3^– NKR-P1⁺ cells (Fig. 4E). Finally, the lack of NK lysis of the Rat2-RT1-E^u/A1ⁿ double transfectant (Fig. 4F) compared with the RT1-E^u transfectant indicated that the expression of RT1-A1ⁿ (self-MHC class Ia) protected the targets from NK cytolysis. Peritoneal cells from naive BN rats were not cytolytic against any of the target cells tested (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Cytolytic activity enriched CD3+ and CD3– NKR-P1+ peritoneal cells from BN rats immunized with WF cells against RT1-transfected Rat2 cells. WF (RT1u) (A), Rat2 cells (B), Rat2 cells transfected with RT1-Au (C), RT1-Eu (D), RT1-A1n (E) or RT1-Eu and RT1-A1n (RT1-Eu/A1n) (F) molecules were used as target cells. The mean ± SD of three experiments is shown.

 
To investigate whether the lack of cytolytic activity against Rat2-RT1-E^u/A1ⁿ cells was associated with a direct interaction with the RT1-A1ⁿ molecule, the RT1-A1ⁿ-specific antibody DN2/4 was used. The anti-RT1-A1ⁿ mAb had no effect on the allo NK killing of Rat2- E^u targets or the absence of killing against Rat2-A1ⁿ targets (Fig. 5A and B). However, the cytolytic activity against Rat2-RT1-E^u/A1ⁿ targets was increased when the anti-RT1-A1ⁿ mAb was added (Fig. 5C), showing that the self-RT1-A1ⁿ molecule is an inhibitory target for in vivo primed allo NK recognition.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Cytolytic activity of CD3– NKR-P1+ NK cells from BN rats immunized with WF (RT1u) cells against RT1-transfected Rat2 cells. Rat2-RT1-Eu (A), Rat2-RT1-A1n (B) and Rat2-RT1-Eu/A1n (C), incubated without mAb () or mAb DN2/4 (anti-RT1-A1n, ), were used as target cells. One representative experiment out of two is shown.

 

	Discussion

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Since i.p. immunization of BN (RT1ⁿ) rats with WF (RT1^u) cells (i) increased the proportion of NK cells among peritoneal cells (25), (ii) increased the proportion of proliferating cells in the NK compartment (38) and (iii) activated NK cells, which killed only targets expressing the same MHC haplotype as the immunizing cells (27), we recently suggested that these NK cells should be regarded as capable of adapting their immune response. In this study we show that not only BN (RT1ⁿ) rats, but also rats of the unrelated strain F344 (RT1^lv1) were capable of inducing allo NK cells when immunized with allogeneic cells, demonstrating selective NK activation to be a general phenomenon in vivo. Since, NK cells are known to lyse allogeneic bone marrow cells, these results, together with others (reviewed in 39,40), underline the potential importance of NK cells in responses to allogeneic bone marrow grafts as well as allogeneic organ grafts (41,42).

We have previously shown that NK cells were activated and selected in vivo via recognition of an allogeneic, non-classical MHC class Ib molecule, RT1-E^u (27). This NK activation was not seen, however, with target cells possessing an allogeneic, classical MHC class I molecule, RT1-A^u. In this study we show that the selective NK cytolysis was inhibited with semiallogeneic targets expressing self-antigens, but not with semiallogeneic targets expressing a third-party alloantigen. These results, together with previous ones showing absence of NK lysis by BN (RT1ⁿ) effectors against RT1ⁿ positive progeny of (WFxBN)xWF backcross (25), showed a correlation between self-MHC expression and protection against NK cytolysis. In addition, we showed that immunization with semiallogeneic cells expressing self-MHC did not activate allo NK cells, which indicates that the initial in vivo `priming' is inhibited by expression of self-MHC (25). In this study we confirm previous results, showing that the NK cells from BN rats (RT1ⁿ) primed in vivo with RT1^u-expressing cells specifically killed targets if they expressed the MHC class I molecule, RT1-E^u. Interestingly, this killing could be inhibited by double-transfecting the target cells with the RT1-A1ⁿ molecule, showing a role for classical self-MHC class I in protection against NK killing. Furthermore, by adding the anti-RT1-A1ⁿ antibody DN2/4 the cytolytic activity against the target cells expressing both the RT1-E^u and RT1-A1ⁿ molecule was recovered. Thus, the protection associated with the expression of the RT1-A1ⁿ molecule was due to a direct interaction between this target ligand and the NK effector cell.

Previous results have shown that when targets expressing or lacking inhibitory MHC class I molecules are present in the same cytolytic assay, targets that lack inhibitory MHC molecules are still lysed by NK cells (43,44). These findings indicate that an inhibitory signal may be most effective when it acts locally to abrogate an activation signal, for instance at the point of contact between an NK cell and its target. The capability of NK cells to respond to cells lacking inhibitory molecules in the presence of cells expressing inhibitory molecules has also been shown in vivo by us. F₁ (WF/BN, RT1^u/n) cells were not able to inhibit the activation of RT1^u-selective allo NK cells, when BN rats were immunized with a mixture of WF (RT1^u) and F₁ (RT1^u/n) cells (results to be published).

Our results that show protection against NK killing by expression of the RT1-A1ⁿ molecule are in accord with the missing-self recognition model, which states that NK cells are inhibited when recognizing self-MHC class I (1 and reviewed in 2). Inhibition of NK self-killing has previously been shown in rat with the allele RT1-A^l (45) and RT1-A^c molecule (J. T. Vaage et al., submitted). The activation of NK cells via recognition of a MHC class Ib molecule and the inhibition of NK via recognition of a self-MHC class Ia molecule, as shown in this study and also suggested by Naper et al. (28 and reviewed in 29), implies that MHC molecules could function as either activation or inhibitory molecules for NK cells. A rat Ly49 NK receptor associated with inhibition when binding to MHC class I has been identified (24,46). However, this receptor was not detected on NK effector cells from BN rats. Although the receptors for the activation and inhibitory MHC class I molecules present on the in vivo primed allo NK cells are, so far, unknown, we would predict from our studies that they are expressed on the same NK subpopulation. Furthermore, the inhibition of allo NK cytolytic activity against cells expressing the self-MHC class Ia molecule, RT1-A, together with the allogeneic MHC class Ib molecule, RT1-E, indicated that the inhibitory signal dominates over the activation signal.

The existence of NK inhibitory receptors recognizing self-MHC class Ia is the mechanism protecting healthy cells of an individual from being killed by activated NK cells. The importance of the capability of the NK cell to kill cells expressing MHC class Ib molecules could hypothetically be evident in certain virus infections, where NK cells have been shown to be involved (47–49). These viruses have been shown to down-regulate MHC class Ia expression. The consequence of this down-regulation may render the virus-infected cells resistant to T cell recognition. However, NK inhibition due to the fact that NK inhibitory receptors recognize self-MHC class Ia molecules will be abrogated. Although not homologous to RT1-E, the human MHC class Ib molecule HLA-E has also been shown to play a significant role in NK cell recognition (50). Interestingly, the HLA-E surface expression was shown to depend on binding of peptides derived from certain HLA class I signal sequences (51). The ability of MHC class Ib molecules to bind viral peptides, however, has not been elucidated. Furthermore, Rolstad et al. have shown activation of NK cytolysis by autologous MHC class Ib in the absence of the self-MHC class Ia (28).

In conclusion, we have identified the self-MHC class Ia allele RT1-A1ⁿ as a molecule protecting targets against allo NK killing. Furthermore, the inhibition via recognition of the self-MHC class Ia molecule, RT1-A, dominates over the activation via recognition of the allogeneic MHC class Ib molecule, RT1-E.

	Acknowledgments

 
We thank E. Gynnstam for technical assistance, Professor T. Gill for providing reagents, and Dr E. Joly for reagents and fruitful discussions. This work was supported by grants from the LEO Research Foundation and Active Biotech Research, Lund. G. W. B. is supported by the UK BBSRC.

	Abbreviations

 

allo NK alloreactive NK

CTL cytotoxic T lymphocyte

	Notes

 
Transmitting editor: H. Wigzell

Received 27 October 1999, accepted 16 February 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Karre, K., Ljunggren, H. G., Piontek, G. and Kiessling, R. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:675.[Medline]
2. Ljunggren, H. G. and Karre, K. 1990. In search of the `missing self': MHC molecules and NK cell recognition. Immunol. Today 11:237.[Web of Science][Medline]
3. Storkus, W. J., Salter, R. D., Alexander, J., Ward, F. E., Ruiz, R. E., Cresswell, P. and Dawson, J. R. 1991. Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2. Proc. Natl Acad. Sci. USA 88:5989.[Abstract/Free Full Text]
4. Karlhofer, F. M., Ribaudo, R. K. and Yokoyama, W. M. 1992. MHC class I alloantigen specificity of Ly-49⁺ IL-2-activated natural killer cells. Nature 358:66.[Medline]
5. Yokoyama, W. M. and Seaman, W. E. 1993. The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. Annu. Rev. Immunol. 11:613.[Web of Science][Medline]
6. Ryan, J. C. and Seaman, W. E. 1997. Divergent functions of lectin-like receptors on NK cells. Immunol. Rev. 155:79.[Web of Science][Medline]
7. Lanier, L. L. 1997. Natural killer cells: from no receptors to too many. Immunity 6:371.[Web of Science][Medline]
8. Lazetic, S., Chang, C., Houchins, J. P., Lanier, L. L. and Phillips, J. H. 1996. Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J. Immunol. 157:4741.[Abstract]
9. Brooks, A. G., Posch, P. E., Scorzelli, C. J., Borrego, F. and Coligan, J. E. 1997. NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor. J. Exp. Med. 185:795.[Abstract/Free Full Text]
10. Carretero, M., Cantoni, C., Bellon, T., Bottino, C., Biassoni, R., Rodriguez, A., Perez-Villar, J. J., Moretta, L., Moretta, A. and Lopez-Botet, M. 1997. The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules. Eur. J. Immunol. 27:563.[Web of Science][Medline]
11. Colonna, M. and Samaridis, J. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268:405.[Abstract/Free Full Text]
12. Wagtmann, N., Biassoni, R., Cantoni, C., Verdiani, S., Malnati, M. S., Vitale, M., Bottino, C., Moretta, L., Moretta, A. and Long, E. O. 1995. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. Immunity 2:439.[Web of Science][Medline]
13. Moretta, A., Bottino, C., Vitale, M., Pende, D., Biassoni, R., Mingari, M. C. and Moretta, L. 1996. Receptors for HLA class-I molecules in human natural killer cells. Annu. Rev. Immunol. 14:619.[Web of Science][Medline]
14. Vivier, E. and Daeron, M. 1997. Immunoreceptor tyrosine-based inhibition motifs. Immunol. Today 18:286.[Web of Science][Medline]
15. Leibson, P. J. 1997. Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity 6:655.[Web of Science][Medline]
16. Moretta, A., Sivori, S., Vitale, M., Pende, D., Morelli, L., Augugliaro, R., Bottino, C. and Moretta, L. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875.[Abstract/Free Full Text]
17. Mason, L. H., Anderson, S. K., Yokoyama, W. M., Smith, H. R., Winkler-Pickett, R. and Ortaldo, J. R. 1996. The Ly-49D receptor activates murine natural killer cells. J. Exp. Med. 184:2119.[Abstract/Free Full Text]
18. Houchins, J. P., Lanier, L. L., Niemi, E. C., Phillips, J. H. and Ryan, J. C. 1997. Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2-C. J. Immunol. 158:3603.[Abstract]
19. Nakamura, M. C., Linnemeyer, P. A., Niemi, E. C., Mason, L. H., Ortaldo, J. R., Ryan, J. C. and Seaman, W. E. 1999. Mouse Ly-49D recognizes H-2D^d and activates natural killer cell cytotoxicity. J. Exp. Med. 189:493.[Abstract/Free Full Text]
20. Lanier, L. L., Corliss, B. C., Wu, J., Leong, C. and Phillips, J. H. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703.[Medline]
21. Lanier, L. L., Corliss, B., Wu, J. and Phillips, J. H. 1998. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693.[Web of Science][Medline]
22. Dissen, E., Berg, S. F., Westgaard, I. H. and Fossum, S. 1997. Molecular characterization of a gene in the rat homologous to human CD94. Eur. J. Immunol. 27:2080.[Web of Science][Medline]
23. Berg, S. F., Dissen, E., Westgaard, I. H. and Fossum, S. 1998. Two genes in the rat homologous to human NKG2. Eur. J. Immunol. 28:444.[Web of Science][Medline]
24. Naper, C., Ryan, J. C., Nakamura, M. C., Lambracht, D., Rolstad, B. and Vaage, J. T. 1998. Identification of an inhibitory MHC receptor on alloreactive rat natural killer cells. J. Immunol. 160:219.[Abstract/Free Full Text]
25. Ericsson, P. O., Hansson, J., Dohlsten, M., Sjogren, H. O., Hiserodt, J. C. and Hedlund, G. 1992. In vivo induced allo-reactive natural killer cells. J. Immunol. 149:1504.[Abstract]
26. Vaage, J. T., Naper, C., Lovik, G., Lambracht, D., Rehm, A., Hedrich, H. J., Wonigeit, K. and Rolstad, B. 1994. Control of rat natural killer cell-mediated allorecognition by a major histocompatibility complex region encoding nonclassical class I antigens. J. Exp. Med. 180:641.[Abstract/Free Full Text]
27. Petersson, E., Holmdahl, R., Butcher, G. W. and Hedlund, G. 1999. Activation and selection of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E. Eur. J. Immunol. 29:3663.[Web of Science][Medline]
28. Naper, C., Rolstad, B., Wonigeit, K., Butcher, G. W. and Vaage, J. T. 1996. Genes in two MHC class I regions control recognition of a single rat NK cell allodeterminant. Int. Immunol. 8:1779.[Abstract/Free Full Text]
29. Rolstad, B., Vaage, J. T., Naper, C., Lambracht, D., Wonigeit, K., Joly, E. and Butcher, G. W. 1997. Positive and negative MHC class I recognition by rat NK cells. Immunol. Rev. 155:91.[Web of Science][Medline]
30. Gonzalez, A. L., Ruffell, D., Butcher, G. W. and Joly, E. 1995. Identification of complementary DNAs for RT1.A(n) and an additional class I molecule in the RT1n haplotype. Transplant. Proc. 27:1516.[Web of Science][Medline]
31. Gunther, E. 1996. Current status of the molecular genetic analysis of the rat major histocompatibility complex. Folia Biol. 42:129.
32. Topp, W. C. 1981. Normal rat cell lines deficient in nuclear thymidine kinase. Virology 113:408.[Web of Science][Medline]
33. Joly, E., Clarkson, C., Howard, J. C. and Butcher, G. W. 1995. Isolation of a functional cDNA encoding the RT1.Au MHC class I heavy chain by a novel PCR-based method. Immunogenetics 41:326.[Web of Science][Medline]
34. Powis, S. J., Young, L. L., Joly, E., Barker, P. J., Richardson, L., Brandt, R. P., Melief, C. J., Howard, J. C. and Butcher, G. W. 1996. The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER. Immunity 4:159.[Web of Science][Medline]
35. Salgar, S. K., Kunz, H. W. and Gill, T., Jr. 1995. Nucleotide sequence and structural analysis of the rat RT1.Eu and RT1.Aw3l genes, and of genes related to RT1.O and RT1.C. Immunogenetics 42:244.[Web of Science][Medline]
36. Hedlund, G., Brodin, T. and Sjogren, H. O. 1987. Selective induction of OX19⁺ (CD5⁺) or OX19^– (CD5^–) alloreactive cytolytic lymphocytes in the rat. Cell. Immunol. 105:366.[Web of Science][Medline]
37. Misra, D. N., Kunz, H. W. and Gill, T. J. d. 1985. Analysis of class I MHC antigens in the rat by monoclonal antibodies. J. Immunol. 134:2520.[Abstract]
38. Petersson, E. and Hedlund, G. 1999. Proliferation and differentiation of alloselective NK cells after alloimmunization-evidence for an adaptive NK response. Cell. Immunol. 197:10.[Web of Science][Medline]
39. Yu, Y. Y., Kumar, V. and Bennett, M. 1992. Murine natural killer cells and marrow graft rejection. Annu. Rev. Immunol. 10:189.[Web of Science][Medline]
40. George, T., Yu, Y. Y., Liu, J., Davenport, C., Lemieux, S., Stoneman, E., Mathew, P. A., Kumar, V. and Bennett, M. 1997. Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts. Immunol. Rev. 155:29.[Web of Science][Medline]
41. Petersson, E., Qi, Z., Ekberg, H., Ostraat, O., Dohlsten, M. and Hedlund, G. 1997. Activation of alloreactive natural killer cells is resistant to cyclosporine. Transplantation 63:1138.[Web of Science][Medline]
42. Petersson, E., Ostraat, O., Ekberg, H., Hansson, J., Simanaitis, M., Brodin, T., Dohlsten, M. and Hedlund, G. 1997. Allogeneic heart transplantation activates alloreactive NK cells. Cell. Immunol. 175:25.
43. Colonna, M., Borsellino, G., Falco, M., Ferrara, G. B. and Strominger, J. L. 1993. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc. Natl Acad. Sci. USA 90:12000.[Abstract/Free Full Text]
44. Nakamura, M. C., Niemi, E. C., Fisher, M. J., Shultz, L. D., Seaman, W. E. and Ryan, J. C. 1997. Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J. Exp. Med. 185:673.[Abstract/Free Full Text]
45. Kraus, E., Lambracht, D., Wonigeit, K. and Hunig, T. 1996. Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition. Eur. J. Immunol. 26:2582.[Web of Science][Medline]
46. Naper, C., Ryan, J. C., Kirsch, R., Butcher, G. W., Rolstad, B. and Vaage, J. T. 1999. Genes in two major histocompatibility complex class I regions control selection, phenotype, and function of a rat Ly-49 natural killer cell subset. Eur. J. Immunol. 29:2046.[Web of Science][Medline]
47. Biron, C. A., Byron, K. S. and Sullivan, J. L. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N. Engl. J. Med. 320:1731.[Web of Science][Medline]
48. Tay, C. H. and Welsh, R. M. 1997. Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells. J. Virol. 71:267.[Abstract]
49. Hengel, H., Brune, W. and Koszinowski, U. H. 1998. Immune evasion by cytomegalovirus—survival strategies of a highly adapted opportunist. Trends Microbiol. 6:190.[Web of Science][Medline]
50. Braud, V. M., Allan, D. S., O'Callaghan, C. A., Soderstrom, K., D'Andrea, A., Ogg, G. S., Lazetic, S., Young, N. T., Bell, J. I., Phillips, J. H., Lanier, L. L. and McMichael, A. J. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795.[Medline]
51. Lee, N., Goodlett, D. R., Ishitani, A., Marquardt, H. and Geraghty, D. E. 1998. HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J. Immunol. 160:4951.[Abstract/Free Full Text]

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