De Novo Donor-Specific HLA Antibody Formation in Two Patients With Crigler-Najjar Syndrome Type I Following Human Hepatocyte Transplantation With Partial Hepatectomy Preconditioning.

Clinical hepatocyte transplantation is hampered by low engraftment rates and gradual loss of function resulting in incomplete correction of the underlying disease. Preconditioning with partial hepatectomy improves engraftment in animal studies. Our aim was to study safety and efficacy of partial hepatectomy preconditioning in clinical hepatocyte transplantation. Two patients with Crigler-Najjar syndrome type I underwent liver resection followed by hepatocyte transplantation. A transient increase of hepatocyte growth factor was seen, suggesting that this procedure provides a regenerative stimulus. Serum bilirubin was decreased by 50%, and presence of bilirubin glucuronides in bile confirmed graft function in both cases; however, graft function was lost due to discontinuation of immunosuppressive therapy in one patient. In the other patient, serum bilirubin gradually increased to pretransplant concentrations after ≈600 days. In both cases, loss of graft function was temporally associated with emergence of human leukocyte antigen donor-specific antibodies (DSAs). In conclusion, partial hepatectomy in combination with hepatocyte transplantation was safe and induced a robust release of hepatocyte growth factor, but its efficacy on hepatocyte engraftment needs to be evaluated with additional studies. To our knowledge, this study provides the first description of de novo DSAs after hepatocyte transplantation associated with graft loss.

Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination.

We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2(d)) and/or C57BL/6J (H-2(b)) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2(d)-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-γ/tumor necrosis factor-α production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12-16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus vaccine compositions.

Evaluation of a new flow cytometry crossmatch procedure for simultaneous detection of cytotoxicity and antibody binding.

In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively. In addition, comparable results were obtained with FCtox IgG and FCXM IgG for both T and B cells. Furthermore, compared with a recently developed and highly sensitive Luminex based C1q assay we obtained close to 90% method agreement with the FCtox assay. Our alternative cytotoxicity and IgG binding assay which exhibit low intra-and inter-assay variation will improve the workflow and speed up the pre-transplant testing and also allow continuous monitoring of assay performance and proper quality assurance.

Detection of complement-fixing and non-fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry.

Donor human leukocyte antigen (HLA)-specific antibodies (Abs) with the ability to activate complement are associated with an increased risk of early Ab-mediated rejection (AMR) of kidney allografts. In recent years, also non-HLA Abs-binding endothelial cells have been shown to elicit early AMR. Donor-specific anti-endothelial cell Abs escape detection in the pre-transplant evaluation if only lymphocytes are used as target cells in crossmatch tests. We addressed whether endothelial precursor cells (EPCs) could be used for detection of complement-fixing as well as non-fixing Abs and if complement factor and immunoglobulin G (IgG) deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent cytotoxicity assay. Deposition of complement factors C3c and C3d, but not C1q nor C4d, were detected on EPCs and lymphocytes upon incubation with HLA Ab-positive sera. There was a correlation between the amount of C3c deposition and IgG binding on EPCs (R(2) = 0.71, P = 0.0012) and T cells (R(2) = 0.74, P = 0.0006) but not for B cells (R(2) = 0.34, P = 0.059). The specificity and sensitivity for C3d deposition on endothelial precursor cell crossmatch (EPCXM) T cells vs the T complement-dependent cytotoxicity (CDC) assay were 69% and 72%, respectively. The EPCXM B-cell C3d assay had considerably lower sensitivity (39%) than the B CDC assay. Altogether, this novel assay based on the detection of complements factors on EPCs and lymphocytes by flow cytometry may widen the diagnostic repertoire and thereby improve the clinical management of patients undergoing kidney transplantation.

A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial precursor cells.

Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8(+) and CD 4(+) T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3(+) lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19(+) lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R(2) = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R(2) = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE assay.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor alpha mediated liver disease.

BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Relation between viral fitness and immune escape within the hepatitis C virus protease.

BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene.

We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8+ CTLs, but was independent of B and CD4+ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6-12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.

Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo.

The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.

Flow cytometric determination of cytokine production and proliferation in hepatitis B core antigen specific murine CD4 cells: lack of correlation between number of cytokine producing cells and cytokine levels in supernatant.

Hepatitis B virus (HBV) core antigen (HBcAg) has extraordinary immunostimulatory properties. The majority of studies done so far on HBcAg induced responses have used ELISA or bioassay for cytokine determination and the 3[H]thymidine incorporation assay to measure proliferation. Here multiparameter flow cytometry was used to measure HBcAg induced cytokine production and proliferation of murine T cells. The advantage with this technique was that we could analyse the cytokine phenotype of proliferating cells of a particular cell type. We found that IL-10 expression was strongly induced in CD4+ T cells after HBcAg immunization. Importantly, we found that IL-4 producing HBcAg-specific CD4+ T cells are common after immunization although detection of IL-4 in culture supernatants indicates only low levels of IL-4. In contrast, IFN-gamma producing HBcAg-specific CD4+ T cells were found at lower numbers despite the detection of high levels of IFN-gamma in culture supernatants. Thus, the frequency of these cells is not accurately reflected by the detectability of the respective cytokine in culture supernatants. A low number of specific CD4+ T cells may effectively produce high levels of cytokine. We therefore suggest that different types of cytokine assays are used in order to obtain the most accurate picture of the intrinsic cytokine phenotype of the CD4+ T cells primed by HBcAg.

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.

External and internal calibration of the MHC class I-specific receptor Ly49A on murine natural killer cells.

Expression of the H-2Dd-specific inhibitory receptor Ly49A on murine NK cells is subject to MHC class I-dependent modulation in vivo. As a result, NK cells in H-2Dd-transgenic mice express low cell surface levels of Ly49A, whereas NK cells from nontransgenic C57BL/6 (B6) mice express high levels. The purpose of this study was to assess the role of MHC class I molecules on the NK cell itself vs those on surrounding cells in this calibration and to test whether the Ly49A levels are subject to regulation in mature NK cells also. Analysis of transgenic mice with mosaic expression of an H-2Dd/Ld transgene showed that MHC class I molecules on surrounding cells (external ligands) and on the NK cell itself (internal ligands) played distinct roles in the determination of Ly49A levels. External ligands were involved in down-regulation of Ly49A levels in vivo, whereas internal ligands kept the down-regulated levels of Ly49A low upon NK cell activation in vitro. Furthermore, in an experimental system based on adoptive transfer of spleen cells, receptor down-regulation of Ly49A occurred as a rapid adaptation process in mature NK cells after interaction with the H-2Dd ligand in vivo. This suggests that Ly49 levels are not fixed but can be changed in mature NK cells when they are exposed to a changed MHC class I environment.

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft.

The MHC class I molecule H-2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered Dd molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A+ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the alpha helix of the alpha2 domain, facing the solvent and without direct contact with the peptide (DdA150S) had no effect. Dd recognition by Ly-49A+ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the Dd molecule that make the trimolecular complex less permissive for inhibition of Ly-49A+ NK cells.

NK cell receptor calibration: effects of MHC class I induction on killing by Ly49Ahigh and Ly49Alow NK cells.

NK cells from C57BL/6 (B6) mice and H-2Dd transgenic B6 (D8) mice express different levels of the inhibitory receptor Ly49A, and they also differ in their target cell specificity. Here, we examined this differential specificity with respect to the role of the Ly49A receptor expression on effector cells and levels of H-2Dd inhibitory ligands on target cells. NK cells from D8 mice express low levels of Ly49A receptor (Ly49Alow), and are able to kill SP2/0 tumor cells in spite of their expression of H-2Dd. H-2Dd is expressed at reduced levels on SP2/0 cells; when these were increased three- to fivefold after IFN-gamma treatment, the killing by Ly49Alow NK cells from D8 mice was markedly reduced. Efficient killing was restored when the effectors were preincubated with anti-Ly49A F(ab')2 Abs. A separate experimental system was based on D8 TAP1-deficient Con A blasts exogenously loaded with H-2Dd-specific peptides. In this system, higher levels of cell surface H-2Dd had to be induced by peptide to inhibit D8 Ly49Alow NK cells to an extent similar to that of B6 Ly49Ahigh NK cells. Ly49A receptors on NK cells from H-2Dd transgenic mice are thus functional, although they require high levels of ligand to inhibit progression of the NK-target cell interaction. The data are in favor of the "receptor-calibration" model, which suggests that down-regulation of inhibitory receptors on NK cells may be useful in order for NK cells to discriminate between normal and reduced levels of MHC class I molecules.

Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice.

MHC class I molecules in the host affect the specificity of NK cells. Previous work has suggested that this specificity is conferred by the expression of products encoded by the Ly49 gene family. This gene family encodes receptors that upon specific recognition of MHC class I ligands mediate an inhibitory signal that prevents killing by NK cells. The pattern of expression of the Ly49 MHC class I binding inhibitory receptors on NK cells is thought to be adapted to the host to ensure the generation of a self-tolerant, yet functional, NK cell repertoire. In the present study we have examined the expression of inhibitory receptors (Ly49A, Ly49C, and Ly49G2) on NK1.1+ cells from B6 (H-2b) and D8 (B6 mice transgenic for H-2Dd) mice as well as corresponding TAP1 -/-, beta2m -/-, and TAP1/beta2m -/- mutants of these mice. We demonstrate that receptor expression on NK1.1+ cells can be specifically modulated by host MHC class I molecules in at least two different ways: alteration of numbers of cells expressing a given receptor and modulation of the levels of expression of a given receptor at the cell surface. The degree of this modulation varies significantly among the various receptors studied and may depend upon the nature of their MHC class I ligands. The results are discussed in relation to the influence of MHC class I molecules on the development of an NK cell repertoire.

Host MHC class I gene control of NK-cell specificity in the mouse.

The missing self model predicts that NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host. Transgenic and gene knock-out mice have provided conclusive evidence that MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the possible mechanisms behind it, starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK-cell functional specificity as well as Ly49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain "useful" NK cells, while avoiding "harmful" and "useless" ones? The available data can be fitted within each of two mutually none-exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as anergy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of beta2-microglobulin.

F1 hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F1 hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2b/d) devoid of beta2-microglobulin (beta2m) are incapable of rejecting beta2m-/- parental C57BL/6 cells (H-2b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F1 mice of the H-2b/d haplotype, requires expression of MHC class I molecules complexed with beta2m.

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice.

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2Dd molecule. The GPI-linked H-2Dd molecule is recognized by H-2Dd-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2Dd molecule on H-2b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2b, H-2Dd) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2Dd molecule as a transgene were able to kill nontransgenic H-2b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.

H-2Dp transgene alters natural killer cell specificity at the target and effector cell levels. Comparison with an H-2Dd transgene.

The expression of MHC class I molecules is an important determinate of natural killer (NK) cell specificity. The missing self hypothesis proposes that NK cells express receptors for self-MHC class I molecules so that target cells that share MHC class I alleles with the NK cells are not killed by those NK cells. However, some effector cells fail to kill some allogeneic target cells suggesting that shared motifs between different MHC class I alleles can interact with the effector cell class I receptors and prevent lysis. We have used transgenic mice to critically assess whether different MHC class I alleles can exert common influences on NK cell specificity at the host/effector and target cell levels. The specificity of NK cells have been compared between C57BL/6 (H-2b) mice and B6DP (H-2b, H-2Dp) and D8 (H-2b, H-2Dd) transgenic mice. The data indicate that H-2Dp and H-2Dd confer similar protection and specific lysis, such that NK cells from either of the H-2Dp or H-2Dd transgenic mice kill nontransgenic target cells yet they do not kill either of the transgenic target cells. The expression of an H-2Dp transgene also provides protection for C57BL/6 lymphoblasts from allogeneic BALB/c (H-2d) NK cells. Furthermore, H-2Dp and H-2Dd transgenic target cells are lysed to a similar extent by H-2k effector cells. These data suggest that H-2Dp and H-2Dd may be able to inhibit the same NK cell population. This may occur through a shared motif recognized by the same receptor, or different motifs recognized by different, but co-expressed receptors.