Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Analysis of the mechanisms involved in NK resistance induced by a new tumor factor NK-RIF

The mechanisms involved in susceptibility or resistance of neoplasic cells to lysis by NK cells are not well known. We have recently described a 12-kDa factor (NK-RIF), produced and released by different tumor cell lines, making K562 resistant to NK lysis without affecting the cytotoxic function of NK effector cells. In this paper we further study the mechanism involved in NK resistance of K562 mediated by NK-RIF and its biological implications. The results show that NK-RIF does not affect the binding capacity of target and effector cells nor the levels of HLA class I antigen expression on the target cells, as a proof that resistance to NK-mediated lysis is not always associated with a defect in target effector binding or with an increased MHC class I antigen expression. However NK-RIF-treated K562 loses its capacity to induce NK cell activation and the subsequent capacity to release NKCF and makes K562 resistant to lysis by NKCF. Therefore our results show that induction of resistance to NK cytotoxicity can be the result of the modulation of target structures responsible for inducing effector cell activation without affecting target/effector binding molecules. This indicates that the structures involved in adherence and activation of NK cells have a different nature and that molecules other than HLA participate in NK resistance.     

Enhanced MHC I antigen expression on tumour target cells is inversely correlated to lysis by allogenic but not by xenogenic NK cells

Relationship between the levels of MHC class 1 antigen expressed on tumour cells and their susceptibility to allogenic and xenogenic NK cells was investigated. Mouse and human natural killer-resistance inducing factor (NK-RIF) preparations were used for augmenting/inducing MHC 1 antigen expression on murine YAC and human K562 tumour cells, respectively YAC cells with augmented MHC I antigen expression became relatively resistant to lysis by murine NK cells but not to rat NK cells. Similarly, induction of MHC I antigens on K562 cells reduced their susceptibility to human NK cells but not to monkey NK cells. These results indicate that the inverse correlation of MHC I antigen expression and NK susceptibility does not hold true for xenogenic pairs of NK effector and target cells.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Inhibition of human NK cell-mediated killing by CD1 molecules

It is now well established that NK cells recognize classical and nonclassical MHC class I molecules and that such recognition typically results in the inhibition of target cell lysis. Given the known structural similarities between MHC class I and non-MHC-encoded CD1 molecules, we investigated the possibility that human CD1a, -b, and -c proteins might also function as specific target structures for NK cell receptors. Here we report that expression of CD1a, -b, or -c can partially inhibits target cell lysis by freshly isolated human NK cells and cultured NK lines. The inhibitory effects of CD1 molecules on NK cell could be shown upon expression of individual CD1 proteins in transfected NK-sensitive target cells, and these effects could be reversed by incubation of the target cells with mAbs specific for the expressed form of CD1. Inhibitory effects of CD1 expression on NK-mediated lysis could also be shown for cultured human dendritic cells, which represent a cell type that prominently expresses the various CD1 proteins in vivo. In addition, the bacterial glycolipid Ags known to be bound and presented by CD1 proteins could significantly augment the observed inhibitory effects on target cell lysis by NK cells.     

Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

Mechanisms involved in NK resistance induced by interferon-gamma.

Human tumor cell lines were treated with interferon-gamma (IFN-gamma) and then used as target cells in NK assays to measure their ability to form conjugates and stimulate the production of NK cytotoxic factors (NKCF) and to determine their susceptibility to NKCF lysis. K562 and cell lines RS1, RS3, RS7, CAC, and CAP2, obtained from solid brain tumors, were used as targets, and peripheral blood lymphocytes (PBL) from normal donors were used as effector cells. IFN-gamma-treated cell lines had a decreased susceptibility to NKCF lysis and a decreased ability to induce the release of these factors without affecting target-effector cell binding. These results were not due to changes in HLA class I antigen expression, given that the level of HLA class I antigens on the tumor cell lines was not affected, the only exception being K562. In an attempt to further clarify the possible influence of HLA class I expression on K562, IFN-gamma-pretreated K562 cells were separated into HLA class I positive and HLA class I negative subsets for the NK assays. The results showed that both populations behaved similarly upon target-effector conjugate formation, whereas the HLA class I positive population showed a reduced susceptibility to lysis by NK cells and NKCF. Thus, these results establish that NK resistance induced by IFN-gamma is mediated by blocking the target cell's ability to activate NK cell triggering and release of NKCF and by blocking its susceptibility to lysis by these factors. This analysis helps to clarify not only the NK process but also the controversial regulatory effect of IFN in NK lysis.     

Influenza virus infection augments NK cell inhibition through reorganization of major histocompatibility complex class I proteins

The killing by natural killer (NK) cells is regulated by inhibitory, costimulatory, and activating receptors. The inhibitory receptors recognize mainly major histocompatibility complex (MHC) class I molecules, while the activating NK receptors recognize stress-induced ligands and viral products. Thus, changes in the expression of the various inhibitory and activating ligands will determine whether target cells will be killed or protected. Here, we demonstrate that after influenza virus infection the binding of the two NK inhibitory receptors, KIR2DL1 and the LIR1, to the infected cells is specifically increased. The increased binding occurs shortly after the influenza virus infection, prior to the increased recognition of the infected cells by the NK activating receptor, NKp46. We also elucidate the mechanism responsible for this effect and demonstrate that, after influenza virus infection, MHC class I proteins redistribute on the cell surface and accumulate in the lipid raft microdomains. Such redistribution allows better recognition by the NK inhibitory receptors and consequently increases resistance to NK cell attack. In contrast, T-cell activity was not influenced by the redistribution of MHC class I proteins. Thus, we present here a novel mechanism, developed by the influenza virus, of inhibition of NK cell cytotoxicity, through the reorganization of MHC class I proteins on the cell surface.     

Immunoproteasome-deficiency has no effects on NK cell education, but confers lymphocytes into targets for NK cells in infected wild-type mice

Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.     

CD66a interactions between human melanoma and NK cells: a novel class I MHC-independent inhibitory mechanism of cytotoxicity

NK cells are able to kill virus-infected and tumor cells via a panel of lysis receptors. Cells expressing class I MHC proteins are protected from lysis primarily due to the interactions of several families of NK receptors with both classical and nonclassical class I MHC proteins. In this study we show that a class I MHC-deficient melanoma cell line (1106mel) is stained with several Ig-fused lysis receptors, suggesting the expression of the appropriate lysis ligands. Surprisingly, however, this melanoma line was not killed by CD16-negative NK clones. The lack of killing is shown to be the result of homotypic CD66a interactions between the melanoma line and the NK cells. Furthermore, 721.221 cells expressing the CD66a protein were protected from lysis by YTS cells and by NK cells expressing the CD66a protein. Redirected lysis experiments demonstrated that the strength of the inhibitory effect is correlated with the levels of CD66a expression. Finally, the expression of CD66a protein was observed on NK cells derived from patients with malignant melanoma. These findings suggest the existence of a novel class I MHC-independent inhibitory mechanism of human NK cell cytotoxicity. This may be a mechanism that is used by some of the class I MHC-negative melanoma cells to evade attack by CD66a-positive NK cells.     

Transfection of beta 2-microglobulin restores IFN-mediated protection from natural killer cell lysis in YAC-1 lymphoma variants

A beta 2-microglobulin (beta 2m)-deficient variant of YAC-1, A.H-2-, was transfected with a genomic beta 2m clone. Transfected cells were used to investigate the role of beta 2m in IFN-induced protection from NK cell lysis. IFN-gamma treatment of the NK-sensitive murine YAC-1 lymphoma results in reduced sensitivity to NK cell-mediated lysis in parallel with increased expression of its constitutively low MHC class I expression. It was previously shown that the A.H-2- variant had lost both these capacities, although it retained other responses to IFN-gamma. Here beta 2m transfection restored the YAC-1 phenotype with respect to an inducible expression of MHC class I molecules and a concomitant protection from NK cell lysis after treatment with IFN-gamma. In the absence of IFN-gamma the NK sensitivity of the transfectants did not differ significantly from A.H-2-. A similar protection from NK cell lysis, in parallel with enhanced MHC class I expression, was observed for in vivo-passaged beta 2m transfectants whereas no protection was found for in vivo-passaged A.H-2- cells. The present study provides evidence that the IFN-gamma-mediated protection from NK cell lysis is dependent on beta 2m expression in the YAC-1 lymphoma. Restoration of MHC class I assembly, transport, and concomitantly an IFN-gamma augmentable cell surface expression of MHC class I molecules is a possible explanation for the effect of beta 2m.     

The regulatory role of CD45 on rat NK cells in target cell lysis.

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

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.     

The switch from latent to productive infection in epstein-barr virus-infected B cells is associated with sensitization to NK cell killing

Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Proteasome inhibitor lactacystin augments natural killer cell cytotoxicity of myeloma via downregulation of HLA class I

Modulation of inhibitory and activating natural killer (NK) receptor ligands on tumor cells represents a promising therapeutic approach against cancer, including multiple myeloma (MM). Human leukocyte antigen (HLA) class I molecules, the NK cell inhibitory killer cell immunoglobulin-like receptor (KIR) ligands, are critical determinants of NK cell activity. Proteasome inhibitors have demonstrated significant anti-myeloma activity in MM patients. In this study, we evaluated the effect of proteasome inhibitors on the surface expression of class I in human MM cells. We found that proteasome inhibitors downregulated surface expression of class I in a dose- and time-dependent manner in MM cell line and patient MM cells. No significant changes in the expression of the MHC class I chain-related molecules (MIC) A/B and the UL16-binding proteins (ULBPs) 1–3 were observed. Downregulation of class I by lactacystin (LAC) significantly enhances NK cell-mediated lysis of MM. Furthermore, the downregulation degree of class I was associated with increased susceptibility of myeloma cells to NK cell killing. HLA blocking antibody produced results that were similar to the findings from proteasome inhibitor. Taken together, our data suggest that proteasome inhibitors, possible targeting inhibitory KIR ligand class I on tumor cells, may contribute to the activation of cytolytic effector NK cells in vitro, enhancing their anti-myeloma activity. Our findings provide a rationale for clinical evaluation of proteasome inhibitor, alone or in combination, as a novel approach to immunotherapy of MM.     

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.     

Synergistic effects of phorbol ester and interferon-alpha: target cell class I HLA antigen expression and resistance to natural killer and lymphokine-activated killer cell-mediated cytolysis

This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-alpha (IFN-alpha) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-alpha or phorbol ester (PDBu). Addition of PDBu together with IFN-alpha had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-alpha and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-alpha made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-alpha. In contrast to PDBu, 4 alpha PDD, a biologically inactive phorbol analogue, alone or combination with IFN-alpha, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class I antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-alpha clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-alpha decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.     

Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity

The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis C virus (HCV) are still unclear. Here, we present evidence for a cascade of molecular events that the virus initiates to subvert the innate immune attack. The HCV core protein induced p53-dependent gene expression of TAP1 (transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I upregulation. Moreover, in p53-deficient liver cell lines, only reconstitution with wild-type p53, but not mutated p53 lacking DNA binding capacity, showed this effect. As a consequence of increased MHC class I expression, a significantly downregulated cytotoxic activity of natural killer (NK) cells against HCV core-transfected liver cells was observed, whereas lysis by HCV-specific cytotoxic T cells was not affected. These results demonstrate a way in which HCV avoids recognition by NK cells that may contribute to the establishment of a chronic infection.