Role of a NK receptor, KLRE-1, in bone marrow allograft rejection: analysis with KLRE-1–deficient mice

Natural killer (NK) cells play a pivotal role in the recognition and rejection of allogeneic target cells, such as bone marrow (BM) allografts.^1-3  NK cell cytotoxicity is exquisitely controlled by signals emanating from stimulatory and inhibitory NK receptors, which recognize major histocompatibility complex (MHC) class I–related molecules on the target cells.^3,4  The function of NK receptor(s) in allograft rejection has been mainly probed with the aid of monoclonal antibodies (mAbs). It is shown that blocking the inhibitory receptor, CD94/NKG2A with anti-CD94 mAb enhances the cytotoxicity of C57BL/6 NK cells against BALB/c concanavalin A (Con A) blasts,⁵  while anti–Ly-49D mAb treatment results in suppression of the BM graft rejection.⁶  In both cases, however, it is still open to question whether the effect of anti-CD94 or anti-Ly49D mAbs was direct or attributed to the cross-reactivity of mAbs with other family members.^5,7-10 

Recently, a novel NK receptor, KLRE-1 (also known as NKG2I), belonging to the killer cell lectinlike receptors (KLRs) family has been characterized.^11-13  A series of experiments using anti–KLRE-1 mAbs indicate that this receptor plays a role in the cytotoxicity mediated by NK cells in vitro.^12,13  However, the definitive assessment for the functions of KLRE-1 has to await KLRE-1–deficient mice.

To this end, we have generated KLRE-1 knock-out mice and assessed the role of KLRE-1. KLRE-1–deficient cells showed little cytolytic activity against allogeneic lymphocytes. Furthermore, allogeneic bone marrow transfer resulted in significant colony formation in the spleen of KLRE-1–deficient mice. These results further confirm that KLRE-1 is a crucial NK receptor for recognition and rejection of bone marrow allograft.

C57BL/6, BALB/c mice were from Charles River Japan (Yokohama, Japan), and 129/svJ mice were from Jackson Laboratory (Bar Harbor, ME). KLRE-1 knock-out mice have been backcrossed 6 times to C57BL/6 mice. Mice were kept under the specific pathogen-free conditions, and 6- to 8-week-old mice were used for the experiments. All experiments were performed in accordance with the guidelines of Chiba University.

KLRE-1 genomic clone was isolated from a mouse genomic library using the KLRE-1 cDNA as a probe (cDNA sequence, first described by Koike et al¹³ ). The genomic DNA fragment from NsiI site located at 1.5 kb upstream of the exon 2 (which contains ATG for first methionine) to BglII site present at 5.0 kb downstream of the exon 2 was used to construct the targeting vector (Figure 1A). Enhanced green fluorescent protein (EGFP)–poly A cassette from the pEGFP-N3 vector (Clontech Laboratories, Palo Alto, CA) was amplified by polymerase chain reaction (PCR) and inserted in frame into the EcoRI site located at 39 bp downstream of ATG, followed by the neomycin-resistant gene (neo^r) cassette flanked by loxP sites. The targeting vector was electroporated into R1 embryonic stem (ES) cells, and G418-resistant clones were screened by Southern blotting with the 5′ external as well as the neo^r internal probes. Positive clones were aggregated with BDF1 blastocysts, and chimeric mice were obtained as described.¹⁴ 

Genotyping was performed by Southern blotting or PCR using genomic DNA from the tail.¹⁴  The probe for Southern blotting was synthesized by PCR with the primer set J03-23 (5′-AAGAGGGAATTCCAGGCACAGATG-3′) and J03-35 (5′-GGGTGCTAAACGGAAATGTAAAGC-3′). The primers used for genotyping were GFP-6 (5′-CCTCTACAAATGTGGTATGGC-3′) and GFP-8 (5′-ATGGTGAGCAAGGGCGAGGAGC-3′) for the targeted allele, and J03KO1 (5′-GATGGATGAAGCACCTGTAAC-3′) and J03KO3 (5′-TCAGAAACCCATCAGACCAACC-3′) for the wild-type allele. The primers for reverse transcriptase (RT)–PCR for KLRE-1 were J03-4 (5′-TAAGAGACAAGCAGGCACGCTGACTG-3′) and J03-7 (5′-ATGGATGAAGCACCTGTAACCCG-3′).

Splenic NK, spleen, bone marrow, and liver mononuclear cells were separated and stained with the appropriate antibodies as described^13,15 ; phycoerythrin (PE)–anti-DX5, cyanin 5 (Cy5)–anti–T-cell receptor β (TCRβ), Cy5–anti-CD3ϵ (PharMingen, San Jose, CA), and Cy-5–labeled antibody against KLRE-1 (anti-NKG2I: 7E8) were used.¹³ 

Cytotoxic assay against the Con A blasts and BM cell transfer experiments were performed essentially as described except that KLRE-1–deficient cells/mice were used in some experiments.¹³ 

We have generated enhanced green fluorescent protein (EGFP)–KLRE-1 knock-in mice to rigorously assess the function of KLRE-1. Southern blot analysis with the 5′ external probe confirmed the correct recombination (Figure 1B). RT-PCR and flow cytometry analyses demonstrated that there was no detectable KLRE-1 transcript or surface expression in the homozygous mice (Figure 1C-D, –/–). A slight decrement of the expression was observed in the heterozygous mice (Figure 1D, left panel, +/–). Concomitantly, the expression of GFP was inversely correlated with the loss of KLRE-1 expression (Figure 1D, right panel). Mice homozygous for EGFP–KLRE-1 (KLRE-1–deficient mice) were born at the expected frequency and were fertile, with no apparent growth abnormality (data not shown). Regarding NK cells, no difference in the profile of CD3/DX5 expression in the spleen, bone marrow (BM), and liver mononuclear cells was noticed between wild-type littermates and knock-out mice (Figure 1E). In addition, no significant variation in the number or ratio of lymphoid subsets was detected in thymus, spleen, and BM cells, suggesting that KLRE-1 is dispensable for the development of lymphoid cells (data not shown).

Previous reports indicate that KLRE-1 plays a pivotal role in allogeneic or redirected lysis.^12,13  Therefore, the roles of KLRE-1 in cytotoxic activity against allogeneic lymphocytes were assessed using KLRE-1–deficient cells. Lymphokine activated killer (LAK) cells from C57BL/6 wild-type mice were mixed with BALB/c Con A blasts, and cytotoxic activity was examined. While wild-type LAK cells showed a significant cytolytic activity against allogeneic target cells, LAK cells from KLRE-1–deficient mice showed little activity (Figure 2A, left panel). In the syngeneic system, however, no marked difference in the cytotoxicity between wild-type and KLRE-1–deficient cells was observed (Figure 2A, right panel). These results demonstrate that KLRE-1 is crucial for NK cells to exert allogeneic cytolytic activity.

We have further explored the function of KLRE-1 in allogeneic BM cell transplantation using KLRE-1–deficient mice (Figure 2B-C). BALB/c BM cells infused to the lethally irradiated C57BL/6 mice were rejected, and no colony formation was seen in the spleen of C57BL/6 mice (Figure 2B-C, WT). In sharp contrast, when BALB/c BM cells were transferred into KLRE-1–deficient mice, a graft dose-dependent colony formation was observed, indicating that the rejection of grafts was severely compromised due to the absence of KLRE-1 (Figure 2B-C, KO). When syngeneic cells were used, the graft was accepted irrespective of KLRE-1 absence and resulted in the formation of a similar number of colonies (Figure 2B, rightmost column). Taken together, one can conclude that KLRE-1 is a crucial mediator for the allograft rejection in vivo.

KLRE-1 has been reported as an inhibitory NK receptor due to its association with the protein tyrosine phosphatase, Src homology domain containing tyrosine phosphatase 1 (SHP-1), and cross-linking of KLRE-1 inhibits NK cell–mediated cytotoxicity.¹²  In our hands, however, KLRE-1 functions as an activating receptor. In fact, cross-linking of NKG2I together with the addition of interleukin-2 (IL-2) and/or IL-12 culminates in the production of interferon γ (IFN-γ).¹³  The inhibition of the cytotoxicity observed in redirected lysis assay may most likely mirror the fact that the absence of KLRE-1 perturbed NK cell–mediated cytotoxicity against allogeneic cells¹²  (Figure 2A, left column). As KLRE-1 is devoid of any signaling motif such as immunoreceptor tyrosine-based inhibitory motif (ITIM) or immunoreceptor tyrosine-based activation motif (ITAM), further works should be necessary to decipher whether KLRE-1 functions as an inhibitory receptor or activating receptor.

In summary, we have shown that KLRE-1 is critical for NK cells to exert allogeneic recognition and rejection using KLRE-1–deficient mice.