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The Journal of Immunology, 2008, 180, 3260-3267
Copyright © 2008 by The American Association of Immunologists, Inc.
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Regulation of the NK Cell Alloreactivity to Bone Marrow Cells by the Combination of the Host NK Gene Complex and MHC Haplotypes1

Koho Iizuka2,*, Anthony A. Scalzo{ddagger},§, Hong Xian* and Wayne M. Yokoyama*,{dagger}

* Department of Medicine, Rheumatology Division and {dagger} Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and {ddagger} Immunology and Virology Program, Centre for Ophthalmology and Visual Science, University of Western Australia and § Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Host NK cells can reject MHC-incompatible (allogeneic) bone marrow cells (BMCs), suggesting their effective role for graft-vs leukemia effects in the clinical setting of bone marrow transplantation. NK cell-mediated rejection of allogeneic BMCs is dependent on donor and recipient MHC alleles and other factors that are not yet fully characterized. Whereas the molecular mechanisms of allogeneic MHC recognition by NK receptors have been well studied in vitro, guidelines to understand NK cell allogeneic reactivity under the control of multiple genetic components in vivo remain less well understood. In this study, we use congenic mice to show that BMC rejection is regulated by haplotypes of the NK gene complex (NKC) that encodes multiple NK cell receptors. Most importantly, host MHC differences modulated the NKC effect. Moreover, the NKC allelic differences also affected the outcome of hybrid resistance whereby F1 hybrid mice reject parental BMCs. Therefore, these data indicate that NK cell alloreactivity in vivo is dependent on the combination of the host NKC and MHC haplotypes. These data suggest that the NK cell self-tolerance process dynamically modulates the NK cell alloreactivity in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer (NK) cells were initially described as having the capability to attack tumor cells without prior sensitization (1). The identification of MHC class I-specific NK cell inhibitory receptors in the early 1990s has revolutionized our conception of how NK cells selectively recognize and attack tumors while sparing normal cells (2, 3, 4). Upon ligand binding, NK inhibitory receptors transmit inhibitory signals to activating receptors. Through an ITIM in its cytoplasmic domain that becomes phosphorylated, inhibitory receptors recruit Src homology 2 domain-containing phosphatase 1, which then cancels out phosphorylation events involved in the NK activation pathways. Significant progress has been made since these initial findings with regard to NK cell inhibitory as well as activating receptors specific to MHC class I or class I-like molecules. This progress has led to the hypothesis that NK cells lacking cognate inhibitory receptors for tumor MHC will have greater activity against those tumor cells. Indeed, in the setting of haploidentical hemopoietic cell transplantation for acute myeloid leukemia, significantly fewer relapses and graft-versus-host disease episodes have been reported in NK receptor-ligand-mismatched donor/recipient pairs than in NK receptor-ligand-matched donor/recipient pairs (5). No correlation between NK receptor-ligand mismatches and relapses has been observed in patients undergoing allogeneic transplant for acute lymphoid leukemia. However, this observation has not been uniformly observed in other transplantation centers (6, 7, 8). Molecular mechanisms of allogeneic MHC recognition by NK receptors have been well characterized at the cellular and molecular levels (9, 10). However, guidelines to understand NK cell alloreactivity under the control of multiple genetic components in vivo remain undetermined and further study with animal models is required to deduce such guidelines.

In contrast to solid tissue transplant rejection that is mediated by components of specific immunity, i.e., T cells, the rejection of allogeneic bone marrow cells (BMCs)3 in lethally irradiated mice is primarily mediated by host NK cells. For example, β2-microglobulin (β2m)-deficient BMCs are readily rejected by wild-type, otherwise syngeneic hosts, indicating a role for MHC class I molecules on donor cells (11). NK cells are responsible for this process because rejection was abrogated by systemic administration of anti-NK cell receptor Abs that deplete NK cells. However, further definition of the NK cell effect is limited with the Ab approach due to the paucity of available mAbs, incomplete description of mAb specificities (12), and expression of multiple receptors by an individual NK cell (13). Furthermore, other host effects are difficult to dissect with this approach. Therefore, unlike the already detailed knowledge of the recognition processes involved in rejection of solid organs, the parameters affecting NK cell rejection of BMC grafts remain to be clearly determined.

Although MHC alleles clearly appear to play some role in bone marrow transplantation (BMT), the genetic transplantation laws governing rejection of BMCs by lethally irradiated mice also differ from those of solid organ transplantation. The most prominent example of these differences is hybrid resistance, whereby F1 hybrid offspring from two H2-disparate strains often reject parental BMCs (14, 15). This phenomenon has been explained by several hypotheses. One suggests that NK cells recognize recessively inherited histocompatibility Ags on parental (donor) BMCs, the Hh-1 (hemopoietic histocompatibility 1) theory (15). To account for the numerous rejection patterns observed in different F1 combinations, it was necessary to presuppose that there must be modifications of the Hh-1 gene expression, further complicating this hypothesis (15, 16, 17). Alternatively, the "missing self"-hypothesis is based on observations that there is an inverse correlation between target cell MHC class I expression and susceptibility to NK cells lysis (18). However, the missing self-hypothesis does not explain all features of hybrid resistance. For instance, F1 hybrid mice sometimes reject one parental bone marrow (BM) graft but not the other, unless T cells are depleted from donor BMCs (19). Hence, there must be other considerations to explain the rules governing BMT with respect to MHC mismatches. "Licensing" is a recently described phenomenon to explain the NK cell tolerance process and functional competence (20). In the licensing process where the ligand-receptor interaction occurs, NK cells gain functional competence, such as killing and cytokine production, and these developmental effects are paradoxically conducted through the ITIM of inhibitory receptors. However, the mechanism of licensing is poorly understood in terms of modes of receptor and ligand interaction, signaling mechanisms and development stages (21). It is not known how the licensing process affects the hybrid resistance.

Allogeneic BMT is a simpler experimental model with which to analyze these complex donor and host effects. The ability to reject allogeneic BMC is recipient strain dependent and donor determinant specific (22). For example, irradiated C57BL/6 (B6, H2b) mice can reject a large inoculum of BMC from H2d mice, whereas irradiated 129 (also H2b) mice do not (recipient strain dependent). By contrast, B6 mice fail to reject a large inoculum of BMCs from H2k haplotype mice (donor determinant specific). Thus, the rejection outcome is frequently unpredictable, when judged only from the perspective of the donor or recipient MHC haplotype (23).

It is formally possible that MHC haplotypes could influence the function of other loci that are involved in BM rejection. One candidate locus was identified using H2b recipients from a backcross panel derived from NK1.1+ (B6) and NK1.1– (129) strains (23). NK1.1 expression was found to be genetically linked to the ability to reject H2d BMCs. However, in backcross panels of H2s recipients (B10.S-H2 s and A.SW), NK1.1 expression did not segregate with rejection of H2ja. Other genetic loci, including host MHC, may have modified the results but this possibility could not be evaluated in the original backcross mice that were sacrificed in the analysis. Nevertheless, in some cases, there was genetic linkage of BM rejection to NK1.1.

The NK1.1 (Nkrp1c) locus resides in the NK gene complex (NKC) on distal mouse chromosome 6 (13). The NKC contains multiple clusters of genes that encode NK cell receptors belonging to the C-type lectin superfamily (24). Many of them recognize MHC class I- or class I-like molecules as ligand. Indeed, transgenic mice expressing inhibitory Ly49 receptors demonstrated their potential role in allogeneic BMT (25, 26), but it was not previously examined in detail how the interaction of a specific NK inhibitory receptor and its ligand regulates functional alloreactivity to third-party BMCs (meaning the allogeneic BMCs that are not recognized by the transgenic NK cell receptor). Moreover, the Ly49 receptors are inherited as haplotypes (27), i.e., clusters of genes that could influence allogeneic BMT. In contrast to Ly49 receptors, Nkrp1 family members do not recognize MHC class I ligands but the C-type lectin-related (Clr) family, which colocalize within Nkrp1 loci (28, 29). With a rat CMV decoy ligand, polymorphisms in the inhibitory Nkrp1b for ligand specificity was recently described (30). Hence, the NKC contains a large number of functional NK cell genes, including NK1.1, and these genes may have different alleles, thus possibly affecting allogeneic BM rejection in a MHC-dependent and -independent manner (31). Although it is theoretically possible to identify specific allotypes of the receptors in the NKC and the MHC class I alleles involved in BMT rejection, as yet, there are not even guidelines to explain the functional interplay of MHC and NKC haplotypes in BMT.

It has not been previously possible to isolate NKC effects or directly examine the influence of the MHC haplotype on the NKC in vivo. In this study, we use NKC and H2-congenic mice in the BMT system to directly assess the effects of NKC haplotypes and the influence of MHC on NK cell alloreactivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6), BALB/c, and SJL/JCr were purchased from the National Cancer Institute (Frederick, MD). C57BL10/J, B10.D2-H2d, B10.S-H2s, B10.WB-H2ja, C.B10-H2b, and SJL/J-B2mtm1Unc were purchased from The Jackson Laboratory. BALB.B6-Cmv1r is a congenic strain in which the murine CMV (MCMV) resistance allele, Cmv1r, as well as other NKC-linked loci from B6 were transferred onto the BALB/c genetic background as described previously (32). Intra-NKC recombinant congenic strains were generated by backcrossing BALB.B6-Cmv1r to BALB/c mice and identifying further intra-NKC recombinants. These strains (BALB.B6-CT3 and BALB.B6-CT6) have smaller segments of the B6-derived NKC region than BALB.B6-Cmv1r as described previously (Ref. 33 and see Fig. 5). C.B10-H2b B6-Cmv1r were generated by sibling mating of (BALB.B6-Cmv1r x C.B10-H2b)F1. Peripheral blood of F2 mice was analyzed by FACS with mAbs specific to H2Kb and H2Dd (AF6-88.5 and 34-5-S, respectively) and tail DNA was genotyped using primers specific for Nkrp1a, Ly49a, and D6Mit13.1 as described previously (34). All mice strains were maintained in a specific pathogen-free barrier facility at Washington University and used at the age of 8–12 wk.

BMT assay

BMT and assessment of engraftment were performed by standard methods as previously described (35). Briefly, after gamma irradiation (9.5 Gy from a 137Cs source) on day 0, recipient mice received the indicated number of BM cells from donor strain via i.v. tail vein injections. On day 5, recipient mice were injected i.v. with 3 µCi of 125I-UdR and 1 x 10–11 mol of FUdR. On day 6, the spleens were removed, rinsed with PBS, fixed in 70% ethanol for 3 h, and the radioactivity was counted with a gamma counter. Incorporation of radioactivity into the spleens was used as an index of hemopoietic precursor cell proliferation. Where indicated, mice were treated with 50 µl of the anti-asialo GM1 Ab (anti-ASGM; WAKO) via i.v. 2 days before BMT to remove endogenous NK cell activity. T cell depletion of BMCs was performed with anti-Thy1.2 Ab (HO-13-4; American Type Tissue Collection) and rabbit complement (Cedarlane Laboratories/Accurate Chemical & Scientific). Each group had at least four mice, unless otherwise noted. At least two experiments were performed for each figure unless mentioned in the figure legend.

Statistics

Multiple comparisons within each experiment were conducted. The experiment-wise error rate was held to the {alpha} = 0.05 level by performing a Sidak t test which held the comparison-wise error rate to be 1– (1– {alpha})1/n, where n is the number of comparisons (36, 37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Multiple genetic factors affect allogeneic BMC rejection

Genetic mechanisms governing allogeneic BM rejection are complicated and controlled by multiple genetic factors (Fig. 1). We transplanted 1 x 106 BMCs from the SJL (H2s) strain into H2-congenic mice on the C57BL/10 (B10) and BALB genetic backgrounds. Five days after BMT, we measured the levels of 125I-UdR incorporation in proliferating donor cells. Whereas we observed a clear H2 effect of rejection of SJL BMCs on the B10 background (i.e., B10 (H2b) and B10.D2 (H2d)), we observed no significant H2 effect on the BALB/c background (i.e., C.B10-H2b (H2b) and BALB/c (H2d)). It is difficult to compare mice with the B10 and BALB backgrounds because we are not controlling for genetic factors other than the H2 loci. Because each strain has a different genetic background, the variability of proliferation could be due to genetic dissimilarities in sensitivity to irradiation or the splenic microenvironment required for donor cells to proliferate. Consistent with these notions, we observed that B10 and B6 mice displayed statistically different responses to allogeneic BMCs of DBA/1 mice in our preliminary studies (K. Iizuka and W. M. Yokoyama, unpublished data). To obtain insights into the genetic events regulating allogeneic BM rejection, we set out to analyze the effects of the NKC and H2 haplotypes by using H2 and NKC-congenic mice.


Figure 1
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  		FIGURE 1. Host MHC haplotypes differentially affect rejection of allogeneic BMCs in different genetic backgrounds. The indicated mouse strains were irradiated and received 106 SJL BMCs i.v. Splenic 125I-UdR uptake was measured as an index of bone marrow engraftment as described in Materials and Methods. All recipient groups contained at least five mice. In multiple comparisons (15 comparisons), the difference between B10 and B6 was not statistically significant (t test p value two tail was 0.0048). This experiment was performed once. Similar results were obtained when 106 T cell-depleted SJL BMCs were transplanted (data not shown).

 
NKC allelic differences dominantly regulate allogeneic BMC rejection

To test whether allelic forms of the NKC affect allogeneic BMT rejection, we transplanted BMCs from SJL into irradiated B6, BALB/c, and BALB.B6-Cmv1r mice; the latter strain is a BALB/c strain congenic for the NKC derived from B6 (Table I). SJL BMCs, ranging from 4 x 105 to 2 x 106 were rejected by B6, but relatively poorly rejected by BALB/c recipients (Fig. 2 and data not shown). By contrast, BALB.B6-Cmv1r and (BALB/c x BALB.B6-Cmv1r)F1 mice rejected SJL BMCs well, to a level similar to that of B6 mice (Fig. 2a). Although splenic 125I-UdR uptake after BMT is an accepted assay of BM engraftment (11, 15), it is possible that the enhanced 125I-UdR incorporation was due to differences in capacity of mature T cells in the donor BM to proliferate in response to recipient allotypes. To eliminate this possibility, we transplanted T cell-depleted BMCs (Fig. 2b). T cell-depleted BMCs from SJL proliferated in BALB/c, but failed to do so in both BALB.B6-Cmv1r and (BALB/c x BALB.B6-Cmv1r)F1 mice, an observation identical with that for mice without T cell depletion (Fig. 2a). This strongly suggests that the results were not due to alloreactive donor T cells. Finally, anti-NK cell depletion and transplantation into mice with selective NK cell deficiency (38) demonstrate that the rejection is NK cell dependent (data not shown), consistent with previous observations suggesting that NK cells are responsible for allogeneic BMT rejection (39, 40). Thus, these data indicate that allelic differences in the NKC of the recipients affect BMT outcome and that these differences are dominant.


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  		Table I. Genetic background of mouse strains

 

Figure 2
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  		FIGURE 2. The NKC regulates allogeneic BMC rejection. a, The indicated mouse strains were irradiated and received 106 SJL BMCs i.v. Splenic 125I-UdR uptake was measured. b, Irradiated mice of the indicated strains received 106 T cell-depleted SJL BMCs and bone marrow engraftment was assessed as in Fig. 1. Each mouse group, BALB/c, BALB.B6-Cmv1r, and (BALB/c x BALB.B6-Cmv1r)F1, was compared with each other for statistical analysis. An asterisk indicates a statistically significant difference between the groups designated with bars. Where asterisks and bars are not shown, there was no significant difference.

 
Recipients recognize β2m-associated molecules in BMC

To evaluate the determinants recognized by transplant recipients, we transplanted BMCs from other strains of mice. Since MHC class I molecules are major determinants recognized by NK cells (13), we next transplanted SJL BMCs deficient in β2m and thus lacking MHC class I expression. BALB/c mice rejected β2m-deficient SJL BMCs as well as BALB.B6-Cmv1r and B6 mice did (Fig. 3), even though BALB/c were poor rejectors of SJL BMCs (Fig. 2a). Interestingly, β2m-sufficient SJL recipient mice failed to reject donated β2m-deficient SJL BMCs. This failure has not been previously reported but is consistent with the previously recognized abnormalities of SJL NK cells (41, 42), or NK cells in SJL may possess a dominant MHC-independent inhibitory receptors. More importantly, BALB/c mice retain both the signal transduction machinery for activation and effector mechanisms necessary to reject SJL BMCs, indicating that BALB/c has molecules to recognize and reject SJL BMCs depending on the recognition of transplanted BMCs. Furthermore, these data indicate that the relevant donor epitope resides in β2m-associated molecules, β2m itself, or both.


Figure 3
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  		FIGURE 3. The NKC effect is β2m-dependent. In brief, 106 β2m-deficient SJL (SJL-B2m) BMCs were transplanted into recipient strains as described in Fig. 1. All recipient groups contained at least five mice, except for the SJL-B2m which contained two mice. Groups of BALB/c and BALB.B6-Cmv1r were statistically analyzed as in Fig. 2 and there was no significant difference. This experiment was performed once.

 
Next, we transplanted BMCs from the B10.S-H2s strain, which has the same H2s haplotype as SJL. The rejection pattern of B10.S-H2s BMCs was comparable to that observed with SJL BMCs (Fig. 4a). Again, T cell-depleted BMCs were rejected by each strain in a manner similar to non-T cell-depleted BMCs (Fig. 4b). Thus, it is likely that recipient NK cells recognize H2s class I or class I-like molecules associated with β2m on transplanted BMCs from SJL and that this process is regulated by recipient NKC alleles.


Figure 4
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  		FIGURE 4. Rejection of BMCs from another H-2 haplotype. a, In brief, 106 B10.S-H2s BMCs were transplanted into indicated recipient strains as described in Fig. 1. The B10.S-H2s recipient group contained two mice. b, In brief, 106 T cell-depleted B10.S-H2s BMCs were transplanted into indicated recipient strains. Groups of BALB/c, BALB.B6-Cmv1r and (BALB/c x BALB.B6-Cmv1r)F1 were statistically analyzed as in Fig. 2 and results are depicted as in Fig. 2.

 
Mapping of allogeneic BM rejection to the region between Cd94 and D6Mit25 on chromosome 6

To determine which region of the NKC is responsible for rejection, we evaluated two intra-NKC recombinant congenic strains, BALB.B6-CT3 and BALB.B6-CT6, both with the BALB/c genetic background (Fig. 5a). BALB.B6-CT3 mice have B6-derived genes from the Cd94 through D6Mit25 interval. In contrast, BALB.B6-CT6 mice have B6-derived genes from Nkrp1c (Klrb1c) through Cd69 only. The BALB.B6-CT3 strain rejected SJL BMCs as well as BALB.B6-Cmv1r, whereas the BALB.B6-CT6 strain mice rejected SJL BMCs minimally, comparable to BALB/c mice (Fig. 5b). F1 mice between BALB/c and either BALB.B6-CT3 or BALB.B6-CT6 rejected SJL BMCs similarly to BALB.B6-CT3 or BALB.B6-CT6 parents, respectively (data not shown). Thus, the capacity to reject SJL BMCs maps to the region between Cd94 and D6Mit25, eliminating the previously mapped Nkrp1c locus as being responsible.


Figure 5
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  		FIGURE 5. Mapping of the locus responsible for allogeneic BM rejection to the region between Cd94 and D6Mit25. a, Schematic representation of intra-NKC-congenic mice. A portion of mouse chromosome 6 with relevant markers is shown on top. For each strain, a thick bar indicates the genome derived from B6 mice, whereas a thin line indicates the BALB/c-derived genome. Schema shown below is an enlargement of the NKC region indicating the general location of the recombination break point for each strain. b, In brief, 106 SJL BMCs were transplanted into indicated recipient strains as described in Fig. 1a. Groups of BALB/c, BALB.B6-CT3, BALB.B6-CT6, and BALB.B6-Cmv1r were statistically analyzed as in Fig. 2 and results are depicted as in Fig. 2.

 
H2 differences influence the effect of the NKC on BM rejection

We next examined whether MHC differences in recipient mice could influence the rejection of SJL BMCs. To this end, we performed sibling mating of (C.B10-H2b x BALB.B6-Cmv1r)F1 to produce C.B10-H2b B6-Cmv1r. This strain is homozygous for the H2b haplotype and NKC derived from B6 on the BALB/c genetic background (Table I). As repeatedly shown, BALB.B6-Cmv1r mice rejected SJL BMCs more vigorously than BALB/c mice, demonstrating a strong effect of the NKC allelic difference in recipients with the H2d haplotype (13.2-fold difference between BALB/c and BALB.B6-Cmv1r). In contrast, there was no difference in SJL BMC rejection when the mice had the H2b haplotype (C.B10-H2b B6-Cmv1r and C.B10-H2b) (no statistically significant difference in multiple comparisons; Fig. 6). These data suggest that NKC allelic effects are differentially modulated by the H2 haplotype and further suggest that the combination of H2 and NKC haplotypes is important in determining the outcome of allogeneic BMC rejection.


Figure 6
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  		FIGURE 6. H2 differences influence the NKC effect on BM rejection. In brief, 106 SJL BMCs were transplanted into indicated recipient strains as described in Fig. 1. All recipient groups contained at least five mice, except for the SJL group which contained three mice. Groups of BALB/c, C.B10-H2b, BALB.B6-Cmv1r, and C.B10-H2b B6-Cmv1r were statistically analyzed as in Fig. 2 and results are depicted as in Fig. 2.

 
To explore this phenomenon with other donor MHC haplotypes, we transplanted B10.WB-H2ja (H2ja) into these strains. When BALB/c mice were congenic for either H2b or NKC derived from B6 (C.B10-H2b or BALB.B6-Cmv1r), the rejection level was not different or only minimally altered (no significant difference or 2.5-fold difference, respectively; Fig. 7). In contrast, BALB/c mice that were congenic for both H2b and NKC from B6 (C.B10-H2b B6-Cmv1r) rejected B10.WB-H2ja BMCs most vigorously (7-fold difference compared with BALB/c; Fig. 7). Therefore, the haplotype combination of NKC and H2 determines the level of BMC rejection.


Figure 7
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  		FIGURE 7. The haplotype combination of both NKC and MHC determines the level of BMC rejection. Two x 106 B10.WB-H2ja BMCs were transplanted into indicated recipient strains as described in Fig. 1. All recipient groups contained at least five mice, except for the B10.WB-H2ja group which contained three mice. Groups of BALB/c, C.B10-H2b, BALB.B6-Cmv1r, and C.B10-H2b B6-Cmv1r were statistically analyzed as in Fig. 2 and results are depicted as in Fig. 2.

 
Allelic differences in NKC affect hybrid resistance

In hybrid resistance, F1 mice often reject BMCs from either parental strain, but sometimes F1 mice reject only one parent’s BMCs and accept the other (15). When F1 mice are generated from non-H2-congenic mice, it is difficult to evaluate the influence of other genetic loci. Our data thus far suggest that allelic differences in the NKC have been overlooked previously and may be important in terms of affecting the outcome of hybrid resistance. To test this possibility, BALB.B6-Cmv1r or BALB/c mice were mated with C.B10-H2b to generate F1 mice that differed only with regard to NKC haplotype (see Table I). These F1 hybrid mice were transplanted with C.B10-H2b parental BMCs (Fig. 8). (BALB/c x C.B10-H2b)F1 mice treated with anti-ASGM (to eliminate host NK cells) accept parental BMCs more readily than nontreated F1 mice, indicating that hybrid resistance occurs in this combination although the resistance is relatively weak. In contrast, (BALB.B6-Cmv1r x C.B10-H2b)F1 mice rejected C.B10-H2b BMCs significantly more than (BALB/c x C.B10-H2b)F1 animals (7.6-fold vs 2.9-fold difference compared with anti-ASGM-treated (BALB/c x C.B10-H2b)F1 mice). Thus, allelic differences of the NKC also directly affect the outcome of hybrid resistance.


Figure 8
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  		FIGURE 8. Allelic difference in the NKC affects hybrid resistance. Two x 106 C.B10-H2b BMCs were transplanted into indicated recipient strains of mice. One group of mice was treated with anti-ASGM 2 days before the BMT. Groups of anti-ASGM-treated (BALB/c x C.B10-H2b)F1, (BALB/c x C.B10-H2b)F1, and (BALB.B6-Cmv1r x C.B10-H2b)F1 mice were statistically analyzed as in Fig. 2 and results are depicted as in Fig. 2. This experiment was performed once. Similar results were obtained when 106 BMCs were transplanted (data not shown). When C.B10-H2b BMCs were used, untreated and anti-ASGM1-treated syngeneic (C.B10-H2b) hosts showed comparable outcomes (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we used NKC-congenic mice to show that NKC haplotypes control allogeneic rejection of SJL BMCs in vivo, formally demonstrating that this genetic region controls a complex outcome, successful engraftment or rejection of allogeneic BMCs. Most importantly, the MHC haplotype differentially influenced the effect of NKC haplotype, resulting in altered levels of rejection of allogeneic BMCs. This indicates that MHC does not always affect allogeneic BM rejection because the MHC influence depends on the NKC haplotype. Thus, these data lead to the conclusion that the combination of both NKC and H2 haplotypes determines the level of allogeneic BMT rejection, i.e., the alloreactivity of NK cells in vivo.

The allelic differences in the NKC also affected hybrid resistance. Although it is possible that different NKC loci regulate allogeneic rejection and hybrid resistance, we currently favor the reductionist interpretation that both rejection systems are regulated by the same NKC locus. Further evaluation of specific NKC loci will be needed to resolve this issue. In contrast, the influence of NKC alleles on hybrid resistance may explain, in part, the previous necessity to propose the Hh-1 theory to account for exceptions to rules otherwise determined by donor-derived MHC haplotypes in hybrid resistance. That the recipient MHC could control BM rejection has been evident for decades, especially in hybrid resistance (15, 43). However, previous studies have been focused on the MHC effect as being due to determinant molecules in MHC that are recognized by recipient. Although there is ample evidence that MHC molecules themselves are likely to be recognized directly on BMCs (44), the concept that the MHC effect is also due to its influence on the NKC control sheds important additional light on previous notions of the role of the MHC in BMT outcomes, such as in hybrid resistance. Furthermore, this study is the first to delineate the importance of NKC allotypes and their influence by MHC in NK cell activities in vivo.

In a previous genetic analysis of host factors that influence the outcome of allogeneic BMT, there was linkage to the NK1.1 locus (23). Due to the small number of backcrossed animals previously used and the close proximity of potentially relevant NKC loci, however, it was not possible to more precisely map the relevant locus or consider alternative hypotheses. Our studies made use of congenic and intra-NKC congenic mice that minimized any differences in non-NKC or non-MHC genes. This permitted more precise mapping to the region between Cd94 and D6Mit25, eliminating NK1.1 as being directly responsible for the "good responder status," and allowed evaluation of other host factors, i.e., MHC, in regulating these effects. It is interesting that SJL mice have an apparent defect in rejecting β2m-deficient SJL BMCs, suggesting MHC-independent inhibition of NK cells (31). Nkrp1d was identified as an inhibitory NK cell receptor recognizing Clrb in a MHC-independent and ITIM-dependent manner (28). Although the Nkrp1 locus is not involved in the allogeneic rejection of SJL BMCs by mice with the B10 background, we examined the possibility that overexpression of Clrb on β2m-deficient BMCs can spare the lysis by engaging the Nkrp1d inhibitory receptor on B6 NK cells in a MHC-independent manner. We observed that β2m-deficient B6 progenitor BMCs transduced with Clrb failed to protect from lysis by host B6 NK cells in vivo (data not shown), suggesting that Clrb expression does not affect the rejection of β2m-deficient BMCs. Although Nkrp1bSJL inhibitory receptor was shown to recognize ClrbB6 (29), it is currently not known whether there is an allelic difference in Clrb between SJL and B6 mice. It will be interesting to examine whether Nkrp1bSJL recognizes ClrbSJL on SJL-β2m BMCs to inhibit rejection in vivo.

Several clues are available with respect to the nature of the allospecific NKC locus against H2s. Since the BMT effect maps to the NKC, we postulate that the effect is due to the function of a NK cell receptor. Because the NKC encodes both activation and inhibitory receptors, the NKC effect may be due to a B6-derived activation receptor or a BALB/c-derived inhibitory receptor. In each case, the reciprocal allele for either hypothesized receptor would either be a null allele or encode a receptor that does not recognize the target ligand. The (BALB x BALB.B6-Cmv1r)F1 hybrid results show a dominant B6 phenotype effect and is consistent with a B6 activation receptor. This receptor should be encoded in the Cd94 to D6Mit25 genomic region as shown by the BALB.B6-CT3 mouse which would be postulated to have gained the B6 activation receptor allele. In contrast, the data are also consistent with a BALB/c-derived inhibitory receptor encoded in the same interval. In this case, the BALB.B6-CT3 mouse would have lost the BALB/c inhibitory receptor allele. A BALB/c inhibitory receptor is consistent with the observation that BALB/c mice completely rejected BMCs from SJL-β2m-deficient mice, indicating that they already express an appropriate activation receptor to reject SJL-derived BMCs and that SJL-β2m-sufficient BMCs may deliver a MHC class I inhibitory signal that prevents rejection. Finally, it is possible that a cluster of highly related genes with overlapping functions and specificities may be involved inasmuch as the NKC contains numerous clusters of such genes (24). Many of those clusters contain genes for activating and inhibitory NK cell receptors and it is now generally believed that a balance of activating and inhibitory signals determines whether NK cells kill or do not kill at the single cell level. It therefore remains possible that the NKC effect on BMT is similarly dependent on both types of receptors.

It is obvious that allogeneic BMT has never exerted selective pressure on the evolution of the immune system in mice. This raises the question: what is the biological purpose or meaning of the combination of both NKC and H2 haplotypes in mice? We propose that the observed phenomenon reflects the NK cell self-tolerance process. B10 or B6 mice are fully capable of rejecting BMCs from B10.D2 mice (data not shown and Ref. 26), indicating that NKC derived from B10 mice encodes NK cell receptors capable of killing B10.D2 BMCs. However, when the H2d haplotype is introduced into B10 mice (i.e., B10.D2 mice), they no longer attack B10.D2 BMCs in BMT because they now are recognized as self. This is true for the combination of C.B10-H2b and BALB/c mice (data not shown). In other words, NK cells are educated to be self-tolerant by the H2d haplotype. Because NKC and MHC loci are located on different chromosomes, NK cell self-tolerance first needs to be established depending on the combination of NKC and H2. In an allogeneic BMT setting, this leads to an alteration in the alloreactivity of NK cells mediated also by the NKC haplotype. Thus, we believe that the mechanisms underlying NK cell tolerance are likely to be the same mechanisms at work in establishing the NK cell-differential alloreactivity by the combination of host H2 and NKC haplotypes.

The nature of the self-tolerance mechanism as it applies to allogeneic BMT rejection is not yet clear at the cellular and molecular levels. Is this the same as licensing (20)? It may be the case for the B10 background. Inasmuch as freshly isolated Ly49A-positive NK cells from B10.D2 mice (expressing H2Dd, the ligand for Ly49A) kill target cells better than the ones from B10 mice (20), Ly49A-positive cells may be licensed to kill SJL BMCs in B10.D2 mice but not in B10. In turn, licensing may be an inadequate explanation of observations from the Ly49C-H2b interaction point of view. B10 mice (expressing H2Kb, the ligand for Ly49C) can license peripheral NK cells expressing Ly49C, but readily fail to reject SJL BMCs. Similarly, NK cells licensed by Ly49A in the BALB background are evidently not competent to reject SJL BMCs, despite the fact that BALB/c and C.B10-H2b mice express Ly49ABALB in the same way as Ly49AB10 in B10 and B10.D2 mice, including specificity, function, expression level, and its alteration by H2 haplotypes (45). Whereas it is not known whether Ly49ABALB or Ly49AB6 can license NK cells on the BALB/c background, several possibilities exist why the NK cell subset licensed by Ly49ABALB does not kill SJL BMCs: 1) the NK cell subset licensed by Ly49ABALB does not express a specific activating receptor for SJL or 2) the licensed subset does express a specific inhibitory receptor for SJL. The former possibility is strongly argued by the observation that BALB/c mice are competent to reject β2m-deficient SJL BMCs, inasmuch as the rejection is mediated by mature, thus "licensed" NK cells. Regarding the latter possibility, it is interesting that Ly49AB6 binds to Con A blasts from B10.S mice (H2s) although the binding is weaker than to Con A blasts with the H2d haplotype (46). Alternatively, mechanisms in addition to licensing may exist to regulate the NK cell tolerance process and activity (47). Elucidation of the cellular and molecular mechanism to explain differential effects of NKC haplotypes by MHC allele requires further studies.

Several NKC loci that affect other NK cell functions in vivo were reported, such as Cmv1, Rmp1, Chok, and Nka (48, 49, 50, 51). Our studies demonstrate that the locus controlling allogeneic BM rejection in mice was also mapped to a region that includes the Ly49 cluster. Although the effector mechanisms underlying rejection of BMC in vivo remain to be determined, attempts have been made to translate the in vitro killing of Con A blasts to hybrid resistance or allogeneic rejection of BMC in vivo (52). However, differential effects on NKC alleles by H2 have not been demonstrated in a genetically controlled manner. Regarding the differential NKC effects by H2, the control of MCMV by Cmv1r locus is especially interesting. Cmv1r was identified as Ly49H in the B6 NKC allele (53, 54, 55) that recognizes m157, a MHC class I-like protein encoded by the MCMV genome (56, 57). Further study of MCMV-resistant MA/My mice indicated that Ly49P, which binds to m157, requires a specific H2 haplotype (H2k) for the Ly49P to efficiently control MCMV infection in vivo (58, 59). However, such a MHC effect was not observed in H2-congenic mice on the BALB background (58), indicating that there is a regulation mechanism mediating differential effects of NKC haplotypes by MHC haplotypes in the NK cell control of MCMV infection. The underlining molecular mechanism also remains undetermined.

The NKC is conserved across species with the syntenic region in humans residing on chromosome 12p13, which also encodes multiple activation and inhibitory NK cell receptors with C-type lectin-like structure, such as CD94/NKG2 and NKG2D (10). Where known, the gene order in mice and humans is identical, with the possible exception that humans do not appear to possess a functional Ly49 gene cluster (60, 61). Also, human NK cells express killer Ig-like receptors (KIR)# that function as HLA class I-specific inhibitory receptors, and noninhibitory receptor Ig-like molecules (10). Both types of Ig-like receptors are encoded in a separate genetic region on chromosome 19q13.4 and many allelic sequences have been reported, most of which are orphan receptors (8). Like in the mouse, the NK receptor and ligand MHC class I genes segregate independently on different chromosomes. Given the similarity between mouse NKC and human KIR loci in terms of allelic and haplotype polymorphisms, our observation of the NK cell alloreactivity defined by the combination of H2 and NKC provides potential insight to understand various outcomes in human BMT therapies with haploidentical hemopoietic cell transplantation (5, 6, 7). That is, setting up the haploidentical hemopoietic cell transplantation, in which the donor HLA and KIR alleles are matched and the recipient HLA is not recognized by the donor KIR, may not necessarily make accurate predictions of potential NK cell alloreactivity to induce graft-vs-leukemia effects. NK cell alloreactivity may be determined by the entire KIR haplotype rather than mismatches in just a few of the inhibitory receptor and ligand interactions. Identification of receptor and ligand specificity with all KIR receptors, including allelic polymorphism and the elucidation of molecular mechanisms underlining NK cell tolerance processes in the mouse model, may provide significant information to improve the BMT therapeutic strategy mediated through NK cell receptors.


   Acknowledgments
 
We thank Kim Marlotte and Debra Rateri for expert technical assistance and breeding and Azza Idris and Lawrence Yu for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (to W.M.Y.). K.I. was supported by a fellowship from the Eastern Missouri Chapter of the Arthritis Foundation. W.M.Y. is an investigator of the Howard Hughes Medical Institute. A.A.S. was supported by a National Health and Medical Research Council Senior Research Fellowship. Back

2 Address correspondence to Dr. Koho Iizuka at the current address, Department of Medicine, Center for Immunology and Cancer Center, University of Minnesota, Minneapolis, MN 55455. E-mail address: iizuk001{at}umn.edu Back

3 Abbreviations used in this paper; BMC, bone marrow cell; β2m, β2-microglobulin; BMT, bone marrow transplantation; BM, bone marrow; NKC, NK gene complex; ASGM, asialo GM1 Ab. Back

Received for publication August 1, 2007. Accepted for publication December 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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