The susceptibility to natural killer cell-mediated lysis of HLA class I-positive melanomas reflects the expression of insufficient amounts of different HLA class I alleles

NK cells selectively lyse tumor cells which do not express one or more MHC class I alleles. The ability to discriminate between self normal or tumor cells is due to the expression of MHC class I-specific killer inhibitory receptors (KIR). In the present study we analyzed melanoma cell lines which were highly susceptible to NK cell-mediated lysis in spite of the expression of a complete set of HLA class I alleles. Quantitative analysis of the HLA class I expression using allele-specific monoclonal antibodies (mAb) revealed a down-regulation of all HLA class I molecules. Treatment of melanoma cells with IFN-γ resulted in up-regulation of all HLA class I alleles that was paralleled by the acquisition of resistance to lysis. That resistance to lysis reflected the up-regulation of HLA class I molecules was revealed by the finding that mAb-mediated masking of either KIR or their HLA class I ligands completely restored the melanoma cell lysis. These results were obtained by the use of selected NK cell clones derived either from allogeneic or autologous donors. In addition, similar results were obtained using in vitro expanded autologous NK cell populations. Our data indicate that NK cells can lyse not only melanoma cells which have lost the expression of one or more HLA class I alleles but also cells expressing a decreased amount of class I molecules.     

p40, a novel surface molecule involved in the regulation of the non-major histocompatibility complex-restricted cytolytic activity in humans

Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions (“p40” molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the “inhibitory” receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double-immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti-p40 mAb. p40^bright cells were homogeneously HLA-DR-positive, p40^medium cells were HLA-DR-negative but co-expressed CD56 antigens, while p40^dull cells were all CD3⁺. Anti-p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti-p40 mAb strongly reduced the anti-CD16 mAb-induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti-CD3 or anti-T cell receptor mAb-mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40-induced inhibition of the NK cytotoxicity required optimal cross-linking, as anti-p40 mAb could inhibit the lysis of Fcγ receptor (FcγR)-positive but not of FcγR-negative target cells. In addition, (Fab')₂ fragments of anti-p40 mAb failed to inhibit the lysis of FcγR-positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK-mediated cytolysis.     

HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex

The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell-mediated lysis. The nonclassical HLA class I molecule HLA-G may mediate protection from NK cells. NK cells are known to express a number of HLA class I-specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I-negative 221 cell line either untransfected or transfected with HLA-G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A-negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA-G-specific inhibitory NKR, and that HLA-G-specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94⁺/NKG2A^? phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA-G specificity, thus suggesting the existence of a different, still unknown NKR.     

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain,FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183^?EB6⁺ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4^? C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183⁺/EB6⁺ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and Fc?RI γ chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58⁺ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ-γ homodimers or in the form of ζ-γ heterodimers, while γ-γ homodimers were detectable in low amounts. However, p58⁺ clones displaying a unique association with γ-γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56^lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56^lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58-associated CD3 ζ, chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 ζ chains.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

Inefficient protection of human TAP-deficient fibroblasts from autologous NK cell-mediated lysis by cytokines inducing HLA class I expression

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.     

Autologous cytotoxicity of natural killer cells derived from HIV-infected patients

NK cells recognize target cells with reduced expression of MHC class I molecules. Human immunodeficiency virus (HIV) infection decreases MHC class I on the cell membrane. The aim of this study was to directly evaluate the role and conditions of NK cell effects in HIV seropositive patients ex vivo. Autologous HIV-infected CD4+ T cells were exposed to NK cells recognition. We discovered an increased lysis of the target cells after infection with human immunodeficiency virus-1 (HIV-1). The expression of the HIV-1 nef gene or the combined expression of nef and tat confers NK susceptibility to autologous CD4+ targets. Downregulation of MHC class I but not HLA-C or CD4 correlated with increased recognition by NK cells. The specific recognition is correlated with downregulation of MHC class I molecules on the infected target cells.     

CD45-mediated regulation of LFA1 function in human natural killer cells. Anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3^?CD16⁺ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3^?CD16⁺ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parralel the inhibition mediated (with the same tumor target cells) by the anti-LFAlα mAb TS.1.22 or by the anti-LFA1β mAb TS.1.18. However, different from the anti-LFAl mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3^?CD16⁺ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16⁺ NK cellsT205 orTA218 mAb were found to reduce by 50–70 % the intracellular Ca⁺⁺ ([Ca⁺⁺]i) mobilization induced by anti-LFAlα or anti-LFA1β mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca⁺⁺ mobilization induced by anti-CD 16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFAla mAb (as “triggering targets”). This effect of anti-LFAlα, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFAlα chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.     

Modification of expression of HLA class I in NK target cells.

Several HLA class I alleles have been shown to downregulate the activity of natural killer (NK) cells in a specific way and recent data indicate that some T cells may be downregulated in the same way. In order to control the expression of certain HLA class I antigens on target cells we work with an HLA hemizygous lymphoblastoid cell line, BM28.7. (HLA-A1, B35, Cw4, DR12). The hemizygosity means that one homologous recombination is sufficient to change the HLA phenotype totally. We use vectors with Zeo (analogous to Neo) as positive selection marker and TK as negative selection marker. The aim of the current construction is to delete the expression of Cw4, and subsequently to insert and express Cw3 in the target cells. The same cell is used to make « normal » transfections with muted Cw3 genes in positions 77, 80 and 88. The latter residue influences glycosylation and we have earlier shown that the N glycosylation of cell surface molecules play a non specific role for NK susceptibility. T cells immortalised with herpesvirus saimiri show an NK like activity and these and other T cells will be used as effector cells along with NK cells against the modified target cells.     

Threonine 80 on HLA-B27 confers protection against lysis by a group of natural killer clones

Recognition of major histocompatibility complex (MHC) class I molecules on target cells by natural killer (NK) cells inhibits NK cell-mediated lysis. Although it is known that this inhibitory effect is regulated by MHC polymorphism, the precise structural determinants remain undefined. Based on the capacity of different HLA-C and HLA-B motifs specifically to inhibit cytotoxicity of some NK clones, three different NK cell specificities (NK1, NK2 and NK3) have been described. In this study, the recognition of HLA-B27 by NK clones has been analyzed using C1R cells transfected with different HLA-B27 subtypes as target cells. Cytotoxicity was inhibited by the HLA-B*2705, -B*2701 -B*2703, -B*2704 and -B*2706 alleles, but not by -B*2702. This subtype is distinguished from the other B27 subtypes by the presence of isoleucine instead of threonine at position 80. Direct involvement of this residue was assessed by showing that site-directed mutagenesis of Thr80 to Ile80 in HLA-B*2705 reverted the NK protective effect of HLA-B*2705. Based on these data, we suggest that Thr80 could act as a single residue conferring target cell protection from lysis by a group of NK clones, tentatively designated NK4.     

Specificity of human T lymphocytes expressing a gamma/delta T cell antigen receptor. Recognition of a polymorphic determinant of HLA class I molecules by a gamma/delta clone

Alloreactive clones expressing T cell receptor (TcR) gamma/delta were derived by limiting dilution from CD3+ CD4- CD8- WT31- populations stimulated in allogeneic mixed lymphocyte culture. These clones specifically lysed phytohemagglutinin-induced blast cells bearing the stimulating alloantigens, whereas they had no effect on autologous or allogeneic unrelated target cells. Analysis of the reactivity with monoclonal antibodies (mAb) specific for two different subsets of TcR gamma/delta (BB3 and delta-TCS-1) showed that five out of nine clones were BB3+, whereas the remaining reacted with delta-TCS-1. Therefore, we can conclude that both subsets of TcR gamma/delta+ cells are able to specifically recognize and lyse allogeneic cells. mAb directed against the CD3-TcR gamma/delta molecular complex strongly inhibited the specific cytolytic activity of TcR gamma/delta+ clones, whereas they had no effect on the lysis of the natural killer-sensitive K-562 target cells mediated by the same clones. An alloreactive delta-TCS-1+ clone (LM12) was further characterized for its specificity. LM12 clone had been derived after stimulation in mixed lymphocyte culture against donor M.M. (HLA typing: Aw68, 24; B35, w55; DR1, 7). The analysis of a large panel of phytohemagglutinin-induced target cells revealed that only the HLA-A24+ target cells were lysed. The direct evidence that the A24 molecule represented the restriction element was provided by experiments using A24-transfected murine P815 target cells. Thus, clone LM12 efficiently lysed A24-transfected P815 cells, but not the same cells untransfected or transfected with the Cw3 gene. Therefore, it appears that polymorphic determinants of class I major histocompatibility complex molecules can be the target of TcR gamma/delta+ alloreactive cell recognition.     

Cytotoxic activity of natural killer cells lacking killer-inhibitory receptors for self-HLA class I molecules against autologous hematopoietic stem cells in healthy individuals

Killer-inhibitory receptors (KIR) are receptors for self-HLA class I molecules, which are expressed on natural killer (NK) cells and small subsets of T-lymphocytes. KIR receptors that do not bind to self-HLA class I have been implicated in the pathogenesis of pure red-cell aplasia and other autoimmune diseases. However, NK cells whose inhibitory receptors lack any apparent self-ligand can also be found in healthy individuals. We therefore tested whether these NK cells are capable of exerting cytotoxic activity against autologous CD34(+) hematopoietic precursors. We detected NK cells whose sole inhibitory receptors were CD94/NKG2-A and that had no affinity for autologous HLA-C molecules. In vitro, such cells were able to kill autologous CD34(+) stem cells that expressed MHC class I antigen at a high level in about 50% of the cases of HLA-C group 2 donors. Two individual clones derived from this NK subpopulation were stimulated by autologous HLA-Cw5/6-positive stem cells, but not by allogeneic HLA-Cw7-positive stem cells. Our findings demonstrate the presence of potentially autoreactive natural killer cells in otherwise healthy individuals.     

Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition

The cytolytic activity of human and mouse natural killer (NK) cells is negatively regulated by self major histocompatibility complex (MHC) class I molecules on potential target cells. In the rat, protection by RT1 class I gene products has so far not been formally shown although the complex effects of foreign and self RT1 genes on polyclonal NK cell activity suggest that MHC recognition can have both stimulatory and inhibitory effects. Here we report that the expression of self-MHC class I molecules on target cells strongly inhibits lysis by a long term NK cell line derived from LEW (RT1^l) rats and by LEW NK cells activated by short-term culture in the presence of interleukin-2. This was demonstrated with mouse-rat hybridoma target cells expressing different rat MHC alleles and with mouse tumor target cells transfected with classical (RT1.A^l) and nonclassical (RT1.C^l) rat MHC class I genes. With hybridoma target cells, the strongest reduction in lysis as compared to the parental mouse myeloma line was observed when “self” (LEW) MHC was expressed, while hybridomas expressing other MHC alleles showed less and variable reduction. Transfection of RT1.A^l protected both L-929 fibroblasts and P815 mastocytoma cells from lysis by the NK cell line, while RT1.C^l only protected P815 cells, indicating that additional target cell properties regulate rat NK cell activity.     

CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.     

Multiple natural killer cell-activating signals are inhibited by major histocompatibility complex class I expression in target cells

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggest that specific properties of the target cell other than the absence of class I expression are necessary to activate NK-mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin-2 activated NK cells, differ in their capacity to kill class I-deficient lymphoblast target cells. In general, class I-deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody-dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK-mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I-mediated inhibition.     

A HLA-Cw6 specific single-chain antibody fragment (scFv) recognizing a natural killer cell receptor epitope

Major histocompatibility complex (MHC) class I molecules induce inhibitory signals on natural killer (NK) cells via killer cell immunoglobulin-like receptors (KIR). We recently reported a human single-chain antibody (scFv#1), which recognizes an epitope on HLA-Cw6 (genotype: *0602). Flow cytometry showed scFv#1 binding to HLA-Cw6 (strong) and also to HLA-Cw2, 4, 5 (very weak) but not to HLA-Cw1, 3, 7, 8. The presumptive epitope of the antibody fragment, which includes residues Asn77 and Lys80 was verified by introducing point mutations into HLA-Cw6 encoding cDNAs. Asn77 --> Ser77 (N77S) and Lys80- -> Asn80 (K80N) mutants of Cw6 lost scFv#1 binding capacity whereas an additional mutation at aa position 90 (Asp-->Ala, D90A) did not influence scFv#1 binding characteristics. Since residues 77 and 80 of HLA-C are directly involved in KIR/MHC interaction, we expected the induction of target cell lysis upon addition of scFv#1 when bringing NK and HLA-Cw6 positive cells together. To prove this interference, we performed Cr-release assays, using Cw*0602 and mock-transfected K562 erythroleukemia cells as targets and freshly prepared peripheral blood NK cells as effector cells. scFv#1 appeared to influence KIR on ligand binding and restored lysis at low effector to target (E/T) ratios. Pan HLA class I antibody W6/32 did not show such effects. Taken together scFv#1 binding patterns with mutagenized HLA-Cw6 and Cr-release assays are strong evidence that the scFv#1 epitope on HLA-Cw6 is at or close to the binding site of CD158a.     

Rat interleukin-2-activated natural killer (A-NK) cell-mediated lysis is determined by the presence of CD18 on A-NK cells and the absence of major histocompatibility complex class I on target cells

The precise mechanism by which target cells are recognized and subsequently lysed by interleukin-2-activated natural killer (A-NK) cells is poorly understood. In this study the role of major histocompatibility complex (MHC) class I and adhesion molecules in the recognition and lysis of tumor cells was investigated in a syngeneic Wag rat model. Preincubation of tumor cells with F(ab′)₂ fragments of anti-MHC class I monoclonal antibody (mAb) OX18 strongly enhanced the A-NK cell-mediated lysis. Also normal syngeneic cells such as T cells and A-NK cells became highly sensitive for lysis by A-NK cells after preincubation with mAb OX18. Two other mAb against MHC class I had no effect on lysis of target cells. These data indicate that masking of MHC class I on syngeneic tumor and normal cells by mAb OX18 is sufficient for A-NK cells to recognize target cells as non-self, resulting in lysis. In addition, we found that the presence of mAb against the β2 (CD18)-integrins blocked the lysis of all tumor cell lines by A-NK cells in ⁵¹Cr-release assays, also when target cells were preincubated with mAb OX18. Because of the absence of CD18 on most tumor cells we concluded that a CD18-associated integrin on A-NK cells is essential for lysis of target cells. These results show that in this syngeneic rat model CD18 on A-NK cells together with MHC class I on tumor cells determine A-NK cell-mediated lysis. Furthermore, we hypothesize that the anti-MHC class I OX18 recognizes an epitope on rat MHC class I which is, or is very close to, the restriction element determining A-NK cell-mediated lysis.     

Identification of NKp80, a novel triggering molecule expressed by human NK cells

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3- and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb-mediated cross-linking of NKp80 resulted in induction of cytolytic activity and Ca2+ mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti-NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb-mediated masking of NKp80 led to a partial inhibition of the NK-mediated lysis of appropriate allogeneic phytohemagglutinin-induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down-regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.