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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Eur J Immunol. 2010 Jun;40(6):1748–1757. doi: 10.1002/eji.200939779

Identification of the Rat NKG2D Ligands, RAE1L and RRLT and Their Role in Allograft Rejection

Ming Zhuo 1, Masato Fujiki 1, Mouer Wang 1, Karine Piard-Ruster 1, Lu-En Wai 1, Liang Wei 1, Olivia M Martinez 1, Sheri M Krams 1,*
PMCID: PMC3057641  NIHMSID: NIHMS268897  PMID: 20306467

Summary

NKG2D is a receptor expressed by natural killer (NK) cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFNγ secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a DAInline graphicLewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day seven post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day one post-transplant. By day seven post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DAInline graphicLewis model, transplantation of Lewis livers into DA recipients (LewisInline graphicDA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in LewisInline graphicDA liver allografts, but significantly increased in rejecting DAInline graphicLewis liver allografts. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

Keywords: NK cells, transplant, liver

Introduction

Natural killer (NK) cells are effectors of the innate immune system that, unlike T cells, do not require prior sensitization to kill target cells. A balance of stimulatory and inhibitory signals derived from membrane receptors regulates NK cell function. Stimulating or activating receptors are characterized by short cytoplasmic tails that lack signaling motifs, but instead generally contain a positively charged residue in the transmembrane domain that associates with immunoreceptor tyrosine-based activating motif (ITAM)-containing signaling adapter molecules, such as CD3ζ, DAP12, and FcεRIγ. NKG2D, a member of the C-type lectin superfamily is a well-characterized stimulatory receptor expressed by NK cells and subsets of T cells [1, 2]. NKG2D delivers an activating signal through association with the DAP10 adapter protein that includes a YxxM motif that serves as an SH2 domain-binding site for the p85 subunit of PI3-kinase [3]. NKG2D interactions with its ligands can activate NK cell effector function including proliferation, cytotoxicity, and cytokine secretion. In humans, two families of NKG2D ligands (NKG2DL) have been reported, the MHC class I related antigen A (MICA), MICB, and members of the UL-16-binding protein (ULBP) family. Rodents lack MIC genes, however NKG2D in mice binds to members of the RAE1 family, the minor histocompatability protein H60 and the murine UL-16-binding protein-like transcript 1 (MULT1). Two sequences of RAE1 family members in rat have been reported, however little functional evaluation of these ligands has been described [4, 5]. In humans and mice, low levels of NKG2DL expression is detected in most normal cells and tissues. Cellular stress, heat shock, viral infection, and transformation all increase the RNA and protein level of NKG2DL [3, 6-8].

Experiments using mouse models of bone marrow transplantation demonstrate the importance of NK cells in the rejection of allogeneic bone marrow [9]. In contrast, NK cells alone are not sufficient to mediate rejection of solid organ allografts since SCID mice, which lack T and B cells but do retain functional NK cells, do not reject allografts [10]. Nevertheless, NK cells have been shown to infiltrate and contribute to rejection of solid organ allografts [11]. Depletion of NK cells results in reduced levels of circulating IFNγ and prolonged survival of liver allografts [12]. NK cells also contribute to the skin graft rejection promoted by CD4+ T cells activated through the indirect allorecognition pathway [13, 14]. Importantly, NK cells, activated by IL-15, are readily able to reject skin allografts in the absence of other lymphocytes indicating that under certain conditions NK cells can mediate graft rejection [14].

Emerging evidence suggests that NKG2D contributes to the rejection of allografts. In bone marrow transplantation, blockade of NKG2D with a neutralizing, non-depleting anti-NKG2D mAb has been shown to prevent NK cell-mediated bone marrow rejection [15]. Similarly, administration of anti-NKG2D mAb prolonged the survival of mouse skin allografts [13]. Further, increased expression of NKG2DL has been reported during allograft rejection in experimental models of murine transplantation [13, 16].

In this study, we have cloned two members of the rat RAE1 family and demonstrate that expression of these two ligands induces effector functions of NK cells. In an experimental model of rat orthotopic liver transplantation, we demonstrate that up-regulation of NKG2DL is observed in rejecting liver allografts but not in liver allografts that survive long term. Our results support a role of NKG2D and its ligands in the rejection of solid organ transplants and suggest that strategies that block NKG2D-NKG2DL interactions may promote graft acceptance.

Results

RAE1L and RRLT are members of the rat RAE1 family

Two members of the rat RAE1 family, RAE1L and RRLT, were cloned from rat lung and testis cDNA respectively. A sequence matching RAE1L is in GenBank, but no functional data has been reported [5]. We cloned a 967 bp PCR product and identified it as RAE1L (Figure 1A). Further, we cloned a 1203 bp PCR product from rat testis cDNA that based on the sequence is another member of the RAE1 family, RRLT (our accession number FJ971664). Similar to what has been described in mice, rat RAE1L and rat RRLT share 89% aa identity, indicating that they both belong to the same gene family. We found that both rat RAE1L and RRLT share only 40% aa identity with mouse RAET1 (Figure 1B). RAE1L and RRLT both contain an MHC class I-like antigen recognition domain predicted by interproscan (http://www.ebi.ac.uk/Tools/InterProScan/), which is also present in all human and mouse NKG2DL.

Figure 1. RAE1L and RRLT are rat homologues of mouse RAET family members.

Figure 1

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(A) Rat RAE1L and RRLT (accession number FJ971664) were cloned and sequenced. The amino acid sequence shares 89% identity when aligned use clustalW although RRLT has an expanded N-terminal region. (B) Both RAE1L and RRLT share ∼40% identity compare to mouse RAE1. Black boxes indicate the same residue while grey boxes indicate a similar residue.

RAE1L and RRLT are expressed at low levels in normal rat tissues

Expression levels of RAE1L and RRLT in normal rat tissues were analyzed by real-time PCR. As RAE1L and RRLT are quite similar in sequence, we generated specific primers for each gene and confirmed that the RAE1L primers did not amplify any product from the RRLT standard sample and, conversely, that the RRLT primers did not amplify any RAE1L products from that standard. RNA was isolated from normal rat tissues and analyzed for both RAE1L and RRLT by real-time PCR (Figure 2). RAE1L and RRLT were each expressed at low levels in heart, lung, skin, spleen, stomach and testis. Neither RAE1L nor RRLT were expressed in normal rat brain, kidney, liver or muscle. RAE1L is expressed at similar levels in both Lewis and DA strains of rat however Lewis rats express higher levels of RRLT. Thus we demonstrate, similar to what has been reported in humans and mice, that rat NKG2DL are absent or expressed at very low levels in normal tissues.

Figure 2. RAE1L and RRLT are absent or expressed at low levels in normal rat tissues.

Figure 2

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The expression of (A) RAE1L (B) and RRLT from normal Lewis (white bars) or DA (dark bars) rat tissue were analyzed by real-time PCR. Data are the mean +/- SEM from three rats in each group and represent the fold increase as compared to the housekeeping gene, G3PDH.

RAE1L or RRLT expressed in CHO cells induces NK cell effector functions

In order to confirm that rat RAE1L and RRLT function as ligands of NKG2D, we generated stable transfectants of RAE1L and RRLT in CHO cells. Both RAE1L and RRLT were detected on the surface of pcDNA3-RAE1L-GFP or pcDNA3-RRLT-GFP transfected CHO cells (Supplementary figure 1). Soluble NKG2D bound to CHO cells transfected with pcDNA3-RAE1L-GFP or pcDNA3-RRLT-GFP further supporting their identity as ligands of NKG2D (Supplementary figure 2). When the NK cell line RNK-16 2B3 or primary rat NK cells were co-cultured with the CHO transfectants expressing RAE1L or RRLT, there was a significant increase in the secretion of IFNγ (Figure 3A). Both RAE1L and RRLT elicited a two-fold increase in IFNγ secretion by primary rat NK cells as compared to control GFP-expressing CHO cells (Figure 3A, black bars) whereas RNK16 NK cells produced three to five fold more IFNγ when cultured with NKG2DL-expressing cells compared to GFP-expressing CHO cells (Figure 3A, white bars). To confirm that NKG2D-NKG2DL interactions were involved in the IFNγ secretion we repeated the co-culture experiment in the presence of our anti-NKG2D mAb (1 ug/ml) or an isotype control antibody. In the presence of anti-NKG2D mAb, IFNγ secretion was significantly decreased, strongly supporting that both RAE1L and RRLT are ligands of NKG2D (Figure 3B).

Figure 3. NK cells produce IFNγ and induce cytotoxicity in response to RAE1L or RRLT.

Figure 3

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(A) RNK16-2B3 cells (white bars) or primary NK cells (dark bars) were co-cultured (1:1) with CHO cells expressing RAE1L or RRLT for 16 hours. (B) RNK16-2B3 cells were co-cultured (1:1) with CHO cells or CHO cells expressing RAE1L or RRLT in the presence of isotype (left) or anti-NKG2D mAb. IFNγ levels were determined by ELISA. Data are the mean±SEM of triplicate wells. RNK16-2B3 cells (C) or primary NK cells (D) (effectors) were mixed with [3H]-thymidine-labeled CHO cells expressing RAE1L (squares) or RRLT (triangles) or GFP (diamonds) (targets) for 8 hr at the indicated E:T ratios. Data are the mean±SEM of triplicate wells. Shown is one representative experiment of three individual experiments. *: P<0.05 (Student's t test).

To determine if RAE1L- and RRLT-expressing target cells could by killed by NK cells, RNK16 cells or primary NK cells were incubated for eight hours at various E:T ratios with RAE1L and RRLT transfected CHO cells and cytotoxicity determined. Both RNK16 and primary rat NK cells readily killed RAE1L- and RRLT-expressing targets (Figure 3CD). At an E:T of 64:1, RNK16 cells effectively killed both RAE1L- and RRLT-expressing CHO cells (45% and 30% killing, respectively as compared to 5% killing of control CHO cells) (Figure 3C). Primary NK cells exhibited more robust killing at a lower E:T ratio with 75% of RAE1L-expressing cells and 55% of RRLT-expressing cells killed at an E:T of 16:1 as compared to 30% killing of control GFP-expressing cells (Figure 3D). These data indicate that NK cells are induced to produce cytokines and kill in response to RAE1L and RRLT and suggest that RAE1L and RRLT are functional ligands of NKG2D.

NKG2D expression is increased in allografts after liver transplantation

To begin to examine the importance of NKG2D-NKG2DL interactions in vivo, a rat model of liver transplantation was utilized. Lewis recipients reject DA livers with a median survival time of 10 days (range 9–12 days). Groups (n=3) of Lewis rats that received Lewis livers (syngeneic) or DA livers (allogeneic) were sacrificed on days one, three and seven post-transplant and the liver graft and spleen were analyzed for expression of NKG2D by real-time PCR. NKG2D expression remains stable in syngeneic grafts at all time points (Figure 4A). In allogeneic liver grafts, there was a ten fold induction of NKG2D at day 3 post-transplant and the levels remain significantly increased through day 7 post-transplant (p<0.05 allogeneic group as compared to syngeneic group). It should be noted that the NKG2D levels in spleen remain stable with no differences detected between the syngeneic group and allogeneic groups (Figure 4B). These data indicate that NKG2D expression is specifically elevated in the allograft.

Figure 4. NKG2D transcript levels are increased in allogeneic liver grafts.

Figure 4

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Expression of NKG2D was determined from tissue obtained from normal rats (gray bars), syngeneic transplants (Lewis->Lewis, white bars) and allogeneic (DA->Lewis, dark bars) liver grafts (A) and spleen (B) on days one, three and seven post-transplant. Data are expressed as the fold induction of NKG2D as compared to the level detected in normal Lewis tissue and represent the mean± SEM of three rats in each group. *: p<0.05 as compared to the syngeneic group (Student's t test).

Transplantation increases NKG2DL expression

To examine the expression of NKG2DL after transplantation, groups (n=3) of Lewis rats that received Lewis livers (syngeneic) or DA livers (allogeneic) were sacrificed on days one, three and seven post-transplant and the liver graft and spleen (day 7 only) analyzed for expression of RAE1L and RRLT using the specific primers we developed and real-time PCR. After transplantation, both RAE1L and RRLT expression are increased in liver grafts (Figure 5A, B). Increased expression of NKG2DL is detected in livers within the first 24 hours post-transplant. Indeed expression levels are similar in both syngeneic and allogeneic livers in the first three days post-transplant. However, on day 7 post-transplant, both RAE1L and RRLT expression were significantly increased (p<0.05 allogeneic group as compared to the syngeneic group), with RAE1L increased 43- fold and RRLT increased 11-fold as compared to normal livers (Figure 5A,B). To determine the source of NKG2DL in transplanted livers, isolated and purified hepatocytes from day 7 livers were analyzed for expression of RAE1L and RRLT. Expression of RAE1L and RRLT were significantly increased in hepatocytes isolated from allografts (DAInline graphicLewis) as compared to hepatocytes from syngeneic (DAInline graphicDA) grafts (Figure 5C). Mononuclear cells isolated from the livers also expressed both NKG2DL but there was no difference between the syngeneic and allogeneic groups (data not shown) suggesting that NKG2DL are increased on hepatocytes during rejection. Interestingly, RAE1L and RRLT levels were not elevated in the spleen post-transplant (Figure 5D) indicating that NKG2D ligands are increased in the transplanted grafts, but not systemically after transplantation.

Figure 5. RAE1L and RRLT expression is increased after transplantation.

Figure 5

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Expression of RAE1L (A) and RRLT (B) from normal rats (gray bars), syngeneic transplants (Lewis->Lewis, white bars) and allogeneic (DA->Lewis, dark bars) liver grafts on days one, three and seven post-transplant. Data are expressed as the fold induction of RAE1L or RRLT as compared to the level detected in normal Lewis tissue and represent the mean± SEM of three rats in each group. (C) RAE1L (light bars) and RRLT (dark bars) expression on purified hepatocytes from liver grants, syngeneic transplants (DA->DA) and allogeneic (DA->Lewis) on day seven post-transplant. Data are expressed as the fold induction of RAE1L or RRLT and represent the mean± SEM of three rats in each group. (D) RAE1L (light bars) and RRLT (dark bars) expression in spleen from syngeneic transplants (Lewis->Lewis) and allogeneic (DA->Lewis) on day seven post-transplant. Data are expressed as the fold induction of RAE1L or RRLT and represent the mean± SEM of three rats in each group. * p<0.05 as compared to the syngeneic group (Student's t test).

Increased expression of NKG2D ligands is associated with allograft rejection

In some strain combinations transplantation of fully histoincompatable livers can result in spontaneous acceptance of the graft. As previously discussed, Lewis rats reject DA livers within two weeks however, in the reverse combination, transplantation of Lewis livers into DA recipients, long-term graft survival spontaneously occurs [17, 18]. The mechanisms that account for these differences in graft outcome have not been determined. NKG2D expression was increased in liver allografts obtained from DA recipients of Lewis livers (spontaneous acceptance model) however the levels of NKG2D were markedly lower than the levels detected in DAInline graphicLewis liver allografts (rejection model) (Figure 6A). To determine if NKG2DL expression was modulated in allografts from the non-rejecting combination, livers from Lewis recipients of DA livers were obtained seven days after transplantation and analyzed for RAE1L and RRLT (Figure 6B,C). In contrast to the rejecting DA—>Lewis livers (far right), NKG2D ligand expression in the non-rejecting allografts Lewis--> DA was similar to the levels detected in syngeneic grafts. Taken together, these data suggest that NKG2D is increased after allogeneic liver transplantation but that increased expression of NKG2DL specifically correlates with rejection of the liver graft.

Figure 6. RAE1L and RRLT expression is increased specifically in rejecting allografts.

Figure 6

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Expression of NKG2D (A) RAE1L (B) and RRLT (C) from normal rats (gray bars), syngeneic liver grafts (white bars) and allogeneic liver grafts (Lewis--> DA, nonrejecting combination and DAInline graphicLewis, rejecting combination, dark bars) seven days post-transplant. Data are expressed as the fold induction of RAE1L or RRLT as compared to the level detected in normal Lewis tissue and represent the mean± SEM of three rats in each group. *: p<0.01(Student's t test).

Discussion

In this study, we cloned two rat NKG2DL, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that expression of these ligands induced IFNγ secretion and cytotoxicity by NK cells. We further demonstrate that expression of the NKG2DL, RAE1L and RRLT, is very low in normal tissues and virtually absent in normal liver, similar to other reports. In particular, we detect low levels of RAE1L and RRLT in normal rat spleen, lung and skin consistent with reports that normal healthy B cells, and epithelium [19] express low levels of NKG2DL. Furthermore, NKG2DL expression is increased in stressed, transformed or pathogen infected cells [2]. DNA-damaging reagents have been shown to induce the expression of NKG2DL [20]. Recent reports indicate that there is both transcriptional and posttranscriptional regulation of NKG2DL as the expression of these ligands must be tightly controlled so as not to promote the destruction of healthy cells [21]. MicroRNAs (miRNA) have been shown to inhibit some ligands of NKG2D. Hcmv-miRUL112A, a miRNA from human cytomegalovirus, has been shown to bind the 3′-UTR of MICB and lead to the downregulation of MICB expression during virus infection [22]. Similarly, several human cellular miRNAs also have the ability to lower the expression of MIC family members in cells [23]. There is also evidence that metalloproteinases induce the shedding of NKG2DL from tumor cells. A recent report suggests that the NKG2DL, MULT 1 is ubiquinated, resulting in its degradation and lack of expression in normal cells [24]. However, “stressful”, events such as UV irradiation and heat shock, reduces ubiquination and thus increases cell surface expression of NKG2DL.

We, and others [11, 12], have demonstrated that activated NK cells link the innate and adaptive immune responses early after transplantation. Specifically we have shown that there is a significant increase in the numbers of host NK cells that infiltrate liver allografts in the first three days post transplant. Depletion of NK cells prolongs liver allograft survival and significantly decreases the levels of IFN-γ after transplantation. Using our novel anti-NKG2D mAb, we show that NKG2D is expressed on both NK cells and T cells isolated from normal, untransplanted rats and from transplanted rats (Supplementary figure 3). Indeed the percentage of NK and T cells from DA rats expressing NKG2D is higher than observed in Lewis rats, in contrast to reports that expression of the Ly-49 receptor is low in DA NK cells [25]. In the current study we demonstrate an increase in the expression of NKG2D in liver allografts (but not in the spleen) in the first week post-transplant. Studies have shown that cardiac allograft survival can be prolonged, in CD28 deficient mice, by administration of antibodies to NKG2D [16]. Further, treatment with both anti-NKG2D antibodies and the fusion protein CTLA4-Ig prolonged cardiac allograft survival in wild-type mice. Thus there is considerable evidence to suggest that NKG2D interactions with their ligands could be important in the rejection of solid organ allografts.

We specifically examined the changes in the NKG2DL RAE1L and RRLT after liver transplantation, and determined that both RAE1L and RRLT were up-regulated in all recipients of liver grafts by the first day post-transplant, suggesting that ischemia/reperfusion injury and surgical trauma induce expression of these ligands. Indeed, RAE1 expression is markedly increased in kidneys during ischemia-reperfusion injury [26] further supporting that stress early after transplant can augment expression of NKG2DL in transplanted solid organ grafts.

Recent reports indicate that NKG2DL expression is increased after transplantation of allogeneic skin and hearts [13, 16]. Similar to our findings, these studies suggest that NKG2DL are increased early in both syngeneic and allogeneic heart grafts, and specifically increased in allografts closer to the time of graft rejection. Isolation of purified hepatocytes and lymphocytes from recipients of syngeneic and allogeneic livers indicate that hepatocytes are the source of the increased expression of the NKG2DL.

In contrast to the increased expression of NKG2DL detected in the transplantation of DA livers into Lewis recipients, transplantation of the reverse combination, Lewis livers into DA recipients, does not result in an increased of RAE1L and RRLT. In fact the levels of these ligands on day seven post-transplant is similar to the levels detected after syngeneic transplant. Although, transplantation of DA livers into Lewis recipients results in acute rejection of the liver allograft, and death of the rat, within two weeks, it is well established that DA recipients of Lewis livers do not reject their livers [17, 18]. DA NK cells have been shown to be deficient in the lysis of allogeneic blasts [27] but it remains unknown if the lack of NK cell alloreactivity has any role in the acceptance of liver allografts. Liver allografts in mice are generally accepted across MHC barriers although the underlying mechanism for this spontaneous tolerance is not clear. Multiple mechanisms including soluble mediators such as MHC class I and Fas, “passenger” lymphocytes transferred with the graft and the presence of liver-derived immature dendritic cells, have been proposed to be responsible for the liver tolerogenicity [3, 28, 29]. A recent report suggests that CD4+, CD25+Foxp3+ T regulatory cells (Treg) are important in the spontaneous tolerance observed in liver allografts in mice [30].

Consistent with this, we have reported that the acquisition of liver tolerance following post-transplant total lymphoid irradiation is associated with apoptosis of graft infiltrating T cells and the subsequent generation of CD4+CD25+Foxp3+ Treg cells [31]. Some recent studies have examined lymphocyte subset and gene expression profiles from operationally tolerant liver transplant recipients in an effort to establish a cellular and molecular profile of tolerance in liver allograft recipients. In one study NK cells were decreased in tolerant recipients as compared to patients on maintenance immunosuppression and normal controls [32] whereas another study detected enrichment in genes encoding for NK cell surface receptors, in tolerant as compared to non-tolerant liver allograft recipients [33]. Additional studies are necessary to clarify the role of NK cells in liver allografts.

Based on the data reported herein we suggest that regulation of NKG2DL on target tissue may be a critical factor in determining NK cell function in the allograft. Therapeutic strategies that block NKG2D interactions with its ligands or prevent the up-regulation of these ligands may prove beneficial in clinical liver transplantation.

Materials and Methods

Cell culture

The rat NK lymphoma line RNK16 and a subline, RNK16-2B3, were maintained in RPMI with 10% fetal bovine serum (FBS) (Serum Source International, Charlotte, NC), 1.0% penicillin/streptomycin (Invitrogen, Carlsbad, CA) and 50 μM 2-mercaptoethanol (Invitrogen) as we have previously reported [34]. Chinese hamster ovary (CHO) cells were maintained in DMEM (Invitrogen) with 10% FBS and 1.0% penicillin/streptomycin. Adherent and suspension 293F cells were grown in DMEM supplemented with 10% FBS and 1.0% penicillin/streptomycin and 4mM L-glutamine (Invitrogen).

Isolation of primary rat NK cells

Primary rat NK cells were isolated and purified from spleens of inbred male Lewis rats as described [35]. Briefly, spleens were removed from Lewis rats and mononuclear cells were isolated by density gradient centrifugation on Ficoll-Hypaque (GE Healthcare, Piscataway, NJ). To prepare purified NK cells, the mononuclear cells were negatively selected using antibodies against CD45A, CD172A (Serotec, Oxford, UK), CD3 and αβTCR (Pharmingen, San Diego, CA) to label B cells, macrophages and T cells, respectively, and then labeled with pan mouse IgG Dynabeads (Dynal, New York, NY). The NK cell-containing fraction was washed, and cultured (0.5×106 /ml) in complete RPMI supplemented with 50 μM 2-mercaptoethanol (Invitrogen), 1.0 mM Na Pyruvate (Invitrogen), 50 mM HEPES (Invitrogen) and 1000 U/ml IL-2 (Biological Resources Branch, National Cancer Institute, Frederick, MD). After culture for a week, >90% of the cells were NKRP1+/CD3- as determined by flow cytometry.

RNA isolation and cloning of RAE1L and RRLT

Total RNA was isolated from 50 mg tissue or 1×106 cells using RNeasy mini plus kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. In the case of tissues, samples were homogenized using a polytron homogenizer. cDNA was generated by using poly T (12-18) primers and superscript reverse trancriptase II (Invitrogen) according to the manufacturer's protocol.

RT- PCR was performed on a PTC 100 thermo-cycler (Bio-Rad, Hercules, CA) using Pfu polymerase (Invitrogen). The RAE1L gene was cloned from cDNA obtained from Lewis rat lung tissue using the following set of primers: sense; 5′-ATGGCAAAGAGAGGAACCACCAAG-3′, antisense; 5′-TCAGCTCCCAGTACTTGAAAACATCTG-3′. The RRLT gene was obtained from the cDNA of Lewis rat heart tissue using the following primers: RRLT sense; 5′-ATGGAGGGTGGAGTTCAGGCTC-3′;. antisense; 5′-TCAGCTCCCAGTACTTGAAAACATCTG-3′. The cycling conditions for amplification were: 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 55°C for 30 s and 72 °C for 2 min. The PCR products were purified by using PCR product purification kit (Qiagen) according to the manufacturer's protocol and cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). Sequencing was performed and both RAE1L and RRLT were fused individually with GFP from pEGFP-C1 (Promega, Madison, WI) and then cloned into pcDNA3 (Invitrogen) for expression.

Expression of RAE1L and RRLT

CHO cells were transfected with pcDNA3-RAE1L-GFP or pcDNA3-RRLT-GFP using FuGENE-6 (Roche, Indianapolis, IN) according to the manufacturer's protocol. After transfection, cells were selected in complete DMEM containing 1.0 mg/ml G418 (Invitrogen) for 1 month. Stable transfected clonal populations of CHO cells uniformly expressing GFP as determined by fluorescence microscopy were used for functional studies.

Cloning of rat NKG2D recombinant protein and antibody production

The extracellular domain of rat NKG2D was fused to the Fc portion of human IgG1, using SOEing PCR (“SOE”: Splicing by Overlapping Extension). A signal peptide was added to the Fc-NKG2D construct by SOEing PCR, in order for the protein to be secreted. The SP-Fc-NKG2D construct was cloned into the pcDNA3.1 expression vector and transfected into adherent 293F cells using FuGENE according to the manufacturer's instructions. The cells were selected in 0.5 mg/ml G418 for 4 days and 0.3 mg/ml G418 for 14 days. Cells were then re-adapted to SFM II medium over the course of one week, and expended in SFM II medium for purification purposes. The recombinant SP-Fc-NKG2D protein was purified on a protein G column (GE healthcare) (Supplementary figure 4). To generate monoclonal antibodies, five mice were immunized and two mice were chosen for cell fusion and hybridoma production. ELISA using human gamma globulin as the antigen confirmed the absence of reactivity against the Fc portion of the recombinant protein. Three hybridoma cell lines were obtained and one cell line, 11D5F4 (IgG1) was chosen for expansion and characterization.

Measurement of IFNγ in culture supernatants

2×105 RNK16 cells or primary NK cells were plated in 24-well plates in complete media. Stable CHO transfectants (2×105) expressing RAE1L, RRLT or GFP were plated in complete RPMI and co-cultured with NK cells for 16hr. In some experiments, 1-5 ug/ml of the anti-NKG2D antibody 11D5F4 was added to cultures of RNK16 cells and RAE1L, RRLT or GFP transfectants. Culture supernatants were then harvested and IFNγ levels determined by ELISA using a rabbit anti-mouse/rat IFNγ coating antibody and a mouse antimouse/ rat IFNγ-biotin (clone DB-1) antibody (Biosource International, Camarillo, CA) as previously described [12]. ELISA data was reported as the mean (pg/ml) of the triplicate +/- SD.

Cytotoxicity assays

Effector cells were plated as two-fold serial dilutions in triplicate in 96-well round-bottom plates. Target GFP, RAE1L or RRLT expressing CHO cells were cultured in complete DMEM and labeled with [3H] thymidine (5 μCi/mL, Perkin-Elmer, Foster City, CA) for 18 h [36]. Washed target cells were plated with or without effector cells for 8 h at 104 target cells/well. Cells were harvested onto a glass fiber filtermat (Wallac, Turku, Finland) using a Tomtec harvester 96MACHII (Orange, CT) and a Wallac1205 betaplate reader and radioactivity measured. Percent specific killing was calculated as follows: (cpm of spontaneous killing without effector cells – cpm of experimental killing) / (cpm of spontaneous killing) × 100.

Animals

Inbred male Dark Agouti (DA) rats and Lewis rats were purchased from Harlan (Indianapolis, IN). All animals were housed, and all procedures were performed, in accordance with a protocol approved by the Stanford Institutional Animal Care and Use Committee.

Orthotopic Liver Transplantation

For syngeneic transplants DA rats received DA livers, or Lewis rats received Lewis livers. Allogeneic transplants consisted of Lewis recipients of DA livers (rejection model) or DA recipients of Lewis livers (non-rejection model). Donor and recipient surgeries were carried out under anesthesia with isoflurane (Abbott Laboratories, North Chicago, IL). Orthotopic liver transplantation, using a modification of Kamada and Calne's technique [37] was performed as we have previously described [12, 38, 39]. Briefly, the liver was perfused with 15 ml of lactated Ringer's solution at 4°C through the catheter placed in the abdominal aorta, and the excised graft was stored in lactated Ringer's solution at 4°C. Cold ischemic time was ≤90 min. Upon completion of the recipient's hepatectomy, the graft was transplanted orthotopically. The anhepatic phase was ≤16 min. No immunosuppression was given to the recipient rats in this study.

Specimens

Groups of recipient rats were sacrificed at days 1, 3, and 7 post-transplant. Liver and spleen tissue were snap frozen for mRNA analysis. When indicated, a portion of the liver graft was utilized for the isolation of liver infiltrating mononuclear cells (LIMC) and hepatocytes.

Mononuclear cell and hepatocyte preparation

Isolation of LIMC was performed as previously described [12]. Briefly, residual blood was removed from the liver by perfusion with PBS through the portal vein. A collagenase solution consisting of 10% FBS (Serum Source International), 0.05% collagenase (Sigma, St. Louis, MO), 5000 U DNaseI (Sigma) in Hanks' buffered salt solution (HBSS, Invitrogen) was then used to perfuse the liver and the liver was then incubated for 10 min at room temperature. Liver tissue was minced, incubated for 30 min at 37°C, and filtered through a nylon mesh to produce a cell suspension. This suspension was then centrifuged at 50g for 5 min to obtain hepatocytes.

The hepatocytes were further purified by centrifugation at 100g in a solution containing Percoll (GE healthcare) and HBSS. Primary hepatocytes (>80% viability) and mononuclear cell were counted and immediately used for RNA isolation. Mononuclear cells were isolated by density gradient centrifugation on Ficoll-Hypaque (GE Healthcare).

Quantitative RT-PCR

Quantitative RT-PCR was performed on an MX3000P real-time PCR system (Stratagene) instrument, according to the manufacturer's instructions. The primers used were: NKG2D sense; 5′-TGTTCGAGTCCTTGTTGCAG-3′; anti-sense, 5′-AGCAGGCTGGAATTTTGAGA-3′. RAE1L sense; 5′-CCTCTCCGGTATGAAGGACA-3′; anti-sense; 5′-CCTTAAGTCCTGGCCCAACAG -3′; RRLT sense; 5′-GCATCCTCTATTCACAGCAGC-3′; anti-sense, 5′-CCCTTAAGTCCTGTTTACATC-3′. Rat G3PDH gene was used as a reference gene. The cycling conditions for real-time PCR were: 50°C for 10 min, followed by 40 cycles at 94°C for 30s, 55°C for 30s and 72°C for 1 min. Data were analyzed by using the MX3000P Analysis Software (Stratagene).

Statistical analysis

PCR results are expressed as the mean ± SD. Group comparisons were performed using the Student's t test. Differences of p< 0.05 were considered significant.

Supplementary Material

Supplementary

Acknowledgments

Supported by funds from the National Institutes of Health AI04495 (SMK) and an NIH ARRA supplement AI044095-09S1.

Footnotes

Conflict of Interest: There are no financial or commercial conflicts of interest

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Supplementary Materials

Supplementary
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