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.     

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.     

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.     

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.     

Evidence for the role of class I and class II HLA antigens in the lytic function of a cloned line of human natural killer cells

Monoclonal antibodies with specificity for HLA class I and class II antigens were generated which either inhibit or enhance the lytic activity of a cloned line of human NK cells. These antibodies were obtained from a fusion with spleen cells from mice immunized with NK clone 3.3. They affect the lytic function of that clone at the level of the killer cell; additional evidence suggests that the effect takes place during an early stage of lysis. Immunoprecipitation and cross-clearing experiments using MHC antibodies of known specificity demonstrate the reactivity of mAbs 131 and 164 with HLA class I antigens and mAbs 210 and 273 with HLA class II antigens. Binding studies indicate that these antibodies are probably recognizing nonpolymorphic MHC determinants. Although these antibodies bind to other NK effector cells tested, they have no effect on the lytic function of these bulk NK populations. Preliminary studies, however, indicate that they do affect the NK activity of a proportion of the clones within these bulk populations. To further assess the potential role of HLA gene products in the lytic function of NK3.3, several well-defined anti-MHC antibodies were tested for their effects on NK3.3 function. The anti-HLA class I and class II antibodies could each be grouped into functional categories based on their ability to enhance, inhibit, or not affect the lysis of NK-sensitive targets K562 and MOLT-4 by NK clone 3.3. These results demonstrate, for the first time, a potential involvement of MHC molecules with NK function.     

Selective MHC expression in tumours modulates adaptive and innate antitumour responses

Progress towards developing vaccines that can stimulate an immune response against growing tumours has involved the identification of the protein antigens associated with a given tumour type. Epitope mapping of tumour antigens for HLA class I- and class II-restricted binding motifs followed by immunization with these peptides has induced protective immunity in murine models against cancers expressing the antigen. MHC class I molecules presenting the appropriate peptides are necessary to provide the specific signals for recognition and killing by cytotoxic T cells (CTL). The principle mechanism of tumour escape is the loss, downregulation or alteration of HLA profiles that may render the target cell resistant to CTL lysis, even if the cell expresses the appropriate tumour antigen. In human tumours HLA loss may be as high as 50%, inferring that a reduction in protein levels might offer a survival advantage to the tumour cells. Alternatively, MHC loss may render tumour cells susceptible to natural killer cell-mediated lysis because they are known to act as ligands for killer inhibitory receptors (KIRs). We review the molecular features of MHC class I and class II antigens and discuss how surface MHC expression may be regulated upon cellular transformation. In addition, selective loss of MHC molecules may alter target tumour cell susceptibility to lymphocyte killing. The development of clinical immunotherapy will need to consider not only the expression of relevant CTL target MHC proteins, but also HLA inhibitory to NK and T cells. Received: 20 March 1999 / Accepted: 3 May 1999     

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.     

Lack of correlation between levels of MHC class I antigen and susceptibility to lysis of small cellular lung carcinoma (SCLC) by natural killer cells

Small cellular lung carcinoma (SCLC) cell lines are susceptible to lysis by NK cells. SCLC, normally negative for MHC class I Ag, were rendered positive for HLA-A and -B Ag by two methods: treatment with IFN-gamma or transfection with HLA class I genes. Exposure to IFN-gamma induced high levels of class I Ag and reduced susceptibility to NK-mediated lysis. However, transfection with either HLA-A2, HLA-B27, or HLA-B27 with beta 2m did not result in reduced susceptibility to NK cells. These transfectants expressed amounts of HLA class I Ag comparable to those in IFN-gamma-treated, untransfected cells. Transfection with the beta 2m gene or plasmid alone neither influenced levels of surface class I Ag nor resulted in reduced susceptibility to lysis by NK cells. Thus, the effects of IFN-gamma on NK susceptibility can be dissociated from the induction of class I Ag.     

NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors

NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.     

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.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

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.     

Analysis of mechanisms by which NK cells acquire increased cytotoxicity against class I MHC-eliminated targets

Acid treatment, where cells are exposed to 0.2 M citric acid buffer at pH 3 for 2 min, was described in a previous paper to be a method which specifically eliminates class I MHC antigens from the membrane of viable cells. We applied this method to characterize functional roles of class I MHC antigens on the target cells in NK cell cytotoxicity. When NK target cells, U937, Molt-4, and Raji, were subjected to acid treatment, the treated cells lost their surface class I MHC antigens and became more sensitive to NK cell killing. On the other hand, the susceptibility of K562 cells which initially lacked class I MHC antigens did not significantly change after such treatment. We then examined the mechanism which enables NK cells to become more cytotoxic against class I MHC antigen-eliminated target cells. Single cell binding assay and cold target inhibition assay demonstrated that class I MHC antigen-eliminated target cells did not acquire high binding affinity to NK cells. However, the interaction between NK cells and class I MHC antigen-eliminated targets resulted in a greater increase in production of NKCF-like factor than did the interaction between NK cells and untreated targets. Class I MHC antigen-eliminated targets did not acquire high killer susceptibility to NKCF-like factor. The present study utilizing the acid treatment method confirmed that surface class I MHC antigens on the targets are important immunoregulatory molecules not only for cytotoxic T lymphocytes but also for NK cells and elucidated some of the underlying mechanisms.     

High expression of the c-myc oncogene renders melanoma cells prone to lysis by natural killer cells

NK cells kill a wide variety of tumor cells, but usually leave normal cells intact. It was earlier reported that low class I HLA expression can be one of the factors that render target cells relatively susceptible to NK lysis. In this contribution, we show that in human melanomas the class I HLA expression is down-modulated by high expression of transfected c-myc oncogenes. The extent of down-modulation depended on the level of c-myc expression in a dose-dependent way. Taken together, these data suggested to us that one of the results of high c-myc expression could be the induction of a NK susceptible phenotype in melanoma cells. Therefore, we analyzed the effect of c-myc on NK susceptibility. We have found that high expression of transfected c-myc genes indeed converts the melanoma cell lines from poor into good targets for NK cells. IFN-gamma was used to restore the class I HLA expression of the c-myc transfectants, and the resulting cells showed a decreased NK susceptibility. These results suggest the possibility that the c-myc-induced NK susceptibility is mediated by the reduction of class I HLA expression.     

Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells

Recent evidence has demonstrated that the lytic function of natural killer cells might be regulated by potential target cells through the target cells' expression of cell surface components that are able to inhibit the lytic process. Specifically, it has been shown in many target cell systems that the expression of class I MHC proteins by target cells is inversely proportional to their susceptibility to lysis by NK cells. It has been suggested, therefore, that MHC proteins may act as important negative regulatory elements in the ongoing control of NK cell function. Herein, we examined two closely related murine lymphoma cells (ASL1 and ASL1w), both in terms of their susceptibility to lysis by NK cells as well as their expression of both H-2K and H-2D class I MHC proteins. The results of these studies showed that whereas ASL1 and ASL1w cells differed greatly in their susceptibility to NK cell lysis (ASL1 was much more NK resistant than ASL1w), both expressed high levels of H-2K and D proteins. In contrast to what might have been predicted base on reports from other target cell systems, the more NK susceptible ASL1w cells expressed somewhat higher levels of H-2K Ag than did ASL1 cells. These results indicate that expression of H-2 class I proteins by target cells, in and of itself, is not sufficient to inhibit the lytic activity of murine NK cells.