The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (LIR-1). Our investigations revealed high levels of LIR-1 expression not only on the NK cell line NK-92, but also on myeloma cells (MOLP-8, RPMI8226) as well as on a lymphoblastoid cell line (LBCL; IM-9). Subsequent cytotoxicity assays were designed to show the isolated effects of LIR-1 blocking on either the effector or the tumor side to rule out receptor-receptor interactions. Although NK-92 was shown to be capable of myeloma cell lysis, inhibition of LIR-1 on NK-92 did not enhance cytotoxicity. Targeting the receptor on MM and LBCL did not also alter NK-92-mediated lysis. We come to the conclusion that LIR-1 alone does not directly influence NK-cell-mediated cytotoxicity against myeloma. To our knowledge, this work provides the first investigation of the inhibitory capability of LIR-1 in NK-92-mediated cytotoxicity against MM and the first functional evaluation of LIR-1 on MM and LBCL.
Understanding of NK cell function has undergone a long process since their identification in 1975 [
NK cell receptors are functionally divided into activating and inhibitory receptors. Their main ligands are major histocompatibility complex I (MHC-I) molecules, while some of the receptors can directly recognize specific antigens on bacteria or damaged cells. Mainly three different subclasses of NK-cell receptors (NKRs) can be distinguished.
LIR and killer immunoglobulin-like receptors (KIRs) are type I transmembrane proteins of the immunoglobulin-like receptor superfamily (IgSF). Both recognize classical HLA class I molecules, while LIR can also interact with nonclassical HLA class I and bacteria with low binding affinities [
LIRs are expressed on subsets of NK cells and T cells, as well as on monocytes, B cells, and DC, with the widest distribution for LIR-1 [
LIR-1 is an inhibitory receptor also known as immunoglobulin-like transcript 2 (ILT-2)/CD85j or leukocyte immunoglobulin-like receptor, subfamily B member 1 (LILRB1) [
MM is an incurable disease that is characterized by the clonal proliferation of terminally differentiated plasma cells [
We therefore investigated the influence of LIR-1 on myeloma defeat. Hereby, we studied the effects of LIR-1 blocking of NK-92 as well as on a panel of tumor cell lines including MM. To our knowledge, these experiments provide the first data concerning the influence of isolated LIR-1 inhibition on NK cells with respect to myeloma cell lysis. Moreover, they provide the first functional study of LIR-1 on MM and on other tumor entities, taking into account its broad distribution among tissues.
Unless otherwise stated, all media and supplements were obtained from Life Technologies. Natural killer cell line NK-92 was cultured in alpha-MEM supplemented with Earl’s Salts and L-Glutamine, 12.5% equine serum, 12.5% fetal calf serum, 0.2 mM inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), and 1% PenStrep. Cells were splitted every third day and received 200 U/mL rhIL-2 (CellSystems) with the fresh medium. Myeloma cell line MOLP-8 was cultured in RPMI1640 with 20% FCS and 1% PenStrep while IM-9, RPMI 8226, HL60, and K562 received the same medium and antibiotics but only 10% FCS. COS-7 cells were cultured in DMEM with 10% FCS and 1% PenStrep. JEG-3 was grown in Ham’s F12 with 10% FCS and 1% PenStrep.
Monoclonal antibodies (mAb) were phycoerythrin- (PE-) conjugated CD2 (RPA-2.10, BDPharmingen), CD159a (Z199, Beckman Coulter), CD85j (HP-F1, Beckman Coulter); Pacific Blue-conjugated CD16 (MOPC-21, BD Pharmingen); fluorescein isothiocyanate- (FITC-) conjugated CD25 (B1.49.9, Beckman Coulter) and anti-IgG (goat polyclonal anti-mouse IgG, Abcam); allophycocyanin (APC)-stained CD56 (B159, BD Pharmingen) as well as appropriate isotype controls. Unconjugated anti-HLA-I (HP-1F7) was obtained from Santa Cruz, anti-HLA-G (MEM-G/09) and -E (MEM-E/08) were obtained from Abcam. 7-Amino-Actinomycin D (7AAD, BD Pharmingen) was used to analyze dead cells. 20
As a positive control for western blot experiments, COS-7 cells were transfected with pCMV6-AC vector encoding for LIR-1 (OriGene) or pCMV6-XL5 as a mock control (OriGene), using FuGene HD Transfection Reagent (Promega) [
Western blot was used to analyze expressed surface molecules of effector and target cells [
Cytolysis was determined in 4-hour chromium-release assays (CRAs) according to standard protocols [
Blocking antibodies were anti-NKG2A (CD159a, IgG2b, Z199 BeckmanCoulter) anti-LIR-1 (CD85j, 292319, IgG2b, R&D Systems) and anti-HLA-I (HP-1-F7, IgG1, Santa Cruz), which blocks HLA-A, -B, -C, -E, and -G engagement [
CRA were performed as described above. Heat inactivated human serum (HS) was obtained after informed consent from healthy volunteers. NK-92 cells were preincubated in RPMI1640 with 1% PenStrep and 10% HS for 30 minutes and kept within the same medium during additional 30 minutes of incubation with mAb concentrations of 0.1, 1 and 10
Target cells were incubated with 1
To control for indirect effects, statistical interpretation was done by multivariate Analysis of Variance (ANOVA). In all calculations, specific lysis was defined as the dependent variable. Antibodies, concentrations of the mAb, the targets and the use or no use of F(ab′)2 were defined as independent variables. Wherever appropriate, interdependencies between the variables were taken into account. All calculations were done by SPSS (IBM SPSS Statistics Version 19, Release 19.0.0).
NK-92 was found to express high levels of LIR-1 based on flow cytometric analysis (Figure
Expression of surface antigens. (a). Flow cytometric phenotyping of all cell lines was done with mABs against LIR-1, CD56, CD16, CNKG2A, CD2, and CD25 as shown for NK-92. (b). Surface antigen expression on NK-92 and tumor cell lines. Values given as % positive staining after subtraction of isotype control.
LIR-1 was present on myeloma cells as well as on IM-9, but no target cell line expressed NKG2A. Western blot analysis confirmed the pattern of LIR-1 expression, and the strength of band representation reflected the staining intensity detected by flow cytometry (Figure
LIR-1 expression on NK-92 and target cells. Western blot analysis of LIR-1 expression on NK-92 cells and tumor cell lines. Untreated as well as LIR-1 or mock transfected COS-7 cells negative and positive controls, respectively. After film development, the membrane was stripped and reincubated with ACTB antibodies as a loading control. 4
HLA expression. (a). Representative flow cytometric gating strategy for analysis of HLA expression on all used cell lines (shown for RPMI8226). All cells were stained with anti-HLA-I/-E/-G IgG1, followed by secondary goat anti-mouse IgG1 FITC. (b). HLA class I molecules were detectable on all cell lines except for negative control K562. JEG-3 were strongly positive for HLA-6, while detection on NK92 and HL60 was marginal. HLA-E as the ligand for NK62A lacked on all cell linesexcept on minor staining on IM-9.
The choice of NK-92 and those distinct tumor cell lines instead of primary cells allowed an isolated view on the inhibitory capacities of LIR-1. No increase of cytotoxicity due to blockade of NKG2A could be expected in subsequent blocking assays, for no target cell line expressed the only known NKG2A ligand HLA-E [
Other inhibitory LIRs that can be found on NK cells are only LIR-3 (ILT5) and LIR-8 as well as soluble LIR-4 for which the ligands are not yet detected [
LIR-1 could therefore be considered to be the only known major inhibitory receptor in this context and was expressed at high levels (Figures
Due to these findings, we considered the use of NK-92 and the chosen tumor cell lines as an ideal system to study the discrete influence of LIR-1 on modulation of NK-cell cytotoxicity.
Cytotoxicity of NK-92 against a panel of tumor cell lines was investigated in CRA at different E : T ratios (Figure
NK-92 mediated killing of target cell lines is dependent on applied E : T ratio. CRA revealed a clearly E : T-dependent level of NK-92 mediated cytotoxicity. Results of CRA are represented as means of at least three independent experiments ± SD. Values in the table below are given as % specific lysis. AML cell line HL60 was almost resistant to NK-92 mediated killing. NK-92 cells were stimulated with 200 U/ml IL-2 the day before the assay.
To evaluate the influence of LIR-1 in myeloma cell lysis, mAbs were used to block LIR-1 receptor-ligand interactions. As target cells lacked the expression of the HLA-E molecule, blocking of NKG2A was not expected to alter the results but was conducted as a negative control. No significant increase of tumor cell lysis could be achieved by any of the mAbs despite high concentrations (Figure
Blocking of CD85j and CD159a on NK-92 does not increase specific lysis. Blocking assays were performed as 4-hour CRA. NK-92 were preincubated with human serum and treated with the respective mAb (0.1, 1 and 10
As specific lysis of target cells by NK-92 was found to be independent from mAb concentration and type, results are presented as means of the used concentrations (0.1/1/10
Use of F(ab′)2 does not significantly influence cytotoxicity of NK-92 cells. Statistical analysis (ANOVA) reveals a seemingly protective effect by F(ab′)2 fragments against NK-92 mediated lysis. As influence of the type of antibody and the used concentration were excluded, experimental data were merged. Results were highly significant (untreated ± F(ab′)2/Ab use ± F(ab′)2): IM-9
What first might appear as a protective effect of the applied F(ab′)2 towards a reduced lysis of target cells could also be observed in the untreated sample and must therefore be considered to be a side effect caused by cell culture procedure. Only experiments with K562 were performed at the same day with and without F(ab′)2 and the observed specific lysis of K562 was the same for both experimental rows. This confirms the thesis of culture side effect to be responsible for significant changes in experimental results.
It is possible that LIR-1 influence could not be measured, if a maximum level of NK-activation had already been achieved before blocking of the inhibitory receptor [
As expected, HLA-A, -B, -C, -E, and -G blockade on tumor cells did not show any influence on lysis (Figure
Blocking of LIR-1 and HLA on tumor cells does not lead to increased lysis by NK-92 cells. LIR-1 (CD85j) and HLA class I molecules of the tumor cell lines were blocked as before on the NK-92 cells. After incubation and extended washing, standard CRA were performed. Neither LIR-1 nor HLA blocking showed significant increase of tumor cell lysis. All experiments were performed with the use of F(ab′)2 to prevent ADCC. Statistical analysis performed by ANOVA excluded any significant effect of either isotype or Ab on target cell lysis, compared to untreated samples. Discrete investigation of K562 treatment, performed as a comparison of cumulated values for all Ab-incubated samples with or without F(ab′)2, excluded any influence of additional F(ab′)2 on the results (
Neither antibodies nor F(ab′)2 show toxic effects on NK-92 cells. NK-92 were labeled with 51Cr and incubated with Abs against LIR-1, NKG2A, or IgG2b. Isotype control with (+) or without (−) F(ab′)2 is under the same conditions as the unlabeled effector cells in the blocking experiments. SR is given as mean of four independent experiments for untreated NK cells and as mean of an experimental series with concentration of 10, 1 and 0.1
To rule out toxic effects of mAb, NK-92 were labeled with 51Cr and incubated with the respective mAb in parallel to the conducted experiments. In four independent experiments, no harmful effect of anti-LIR-1, anti-NKG2A or F(ab′)2 could be observed (Figure
Although not significantly affecting tumor lysis, there seemed to be an important influence of the F(ab′)2 fragments (Figure
Use of F(ab′)2 after blocking NK-92 receptors seems to balance lysis pattern. Values show the % relative increase or decrease of target cells lysis in a standard 4-hour CRA, calculated as follows: % relative change of specific lysis = [(% specific lysis “antibody”) − (% specific lysis “untreated”)]/[% specific lysis “untreated”]. Though no concentration of the used mAbs showed a significant change in tumor lysis, interesting differences in the resulting lysis-patterns could be detected. (a) Apart from outliers, use of F(ab′)2 evens out mAb effects in the blocking experiments, leading to values that oscillate close to the origin in both directions. (b) Sparing F(ab′)2 decreases specific lysis, predominantly for MOLP-8 and RPMI8226 cells.
The aim of the present investigations was to evaluate if LIR-1 on NK cells inhibits NK-92 mediated cytotoxicity against different tumor cell lines. Secondly, presence of LIR-1 on the target cells was validated concerning its influence NK cell mediated lysis. LIR-1 was assumed to be the only relevant inhibitory receptor on NK-92 as stated above (characterization of NK-92 and tumor cell lines) [
By now, involvement of LIR-1 in protecting the fetus from abortion has become common immunological knowledge, and so has the adoption of this mechanism by tumor cells by expressing the LIR-1 ligand HLA-G [
Godal et al. made an attempt in revealing the influence of LIR-1 on dNK in cytotoxicity against HLA-G negative AML and ALL blasts. Their results indicate that LIR-1 does only serve as a weak inhibitory NK cell receptor in the absence of HLA-G on tumor cells, but might be relevant in situations with low KIR expression as seen within the first months after stem cell transplantation (SCT) [
Other authors used merely HLA-transfected 721.221, murine cells (P815) or immature dendritic cells (iDC) as target cells, but often had the benefit of comparing results from NK cell lines to the performance of NK cells derived from healthy donors. In 2008, Yawata et al. conducted a series of degranulation assays of dNK against HLA class I deficient 721.221 in order to investigate the involvement of distinct receptors in “missing-self” recognition and were not able to identify any involvement of LIR-1 [
No such conflict was observed by Favier et al., who incubated dNK and the NK cell line NKL with 721.221-G1 and used blocking Abs against LIR-1 and HLA-G [
Moreover, in reverse ADCC (rADCC) assays with dNK against P815, only few clones were inhibited by LIR-1. Here again, NK cell clones have shown to exhibit less predictable outcomes than cell lines [
Summarizing the available data, NK cell lines seem to be more reliable than dNK concerning LIR-1 mediated downregulation of cytotoxicity. The results for polyclonal dNK show high variances between the different clones that are mostly not characterized in detail. Successful LIR-1 mediated inhibition by HLA-transfected 721.221 has abundantly been shown. Involvement of additional undetected receptors could not always be excluded, and different types of assays performed with the same effector and target cells could lead to highly diverging results. Investigations of cytotoxicity of dNK and donor-derived tumor cells are rare and no sufficient information is available about the role of LIR-1 in the absence of HLA-G. Current opinion about the inhibitory influence of LIR-1 is predominantly based on investigations at the feto-maternal interface or has been gained from settings in which only a single HLA molecule was present on the target cells—mostly HLA-G or -B on 721.221. The performed experiments do not sufficiently cover the extensive binding capacities of LIR-1 to HLA class I.
Although we are aware of problems concerning the comparability of NK cell lines to donor-derived NK cells (dNK), we have chosen an experimental setting that allows studying isolated influence of LIR-1 on cytotoxicity. Our major goal was to provide a model system to overcome the common practice of using transfected target cell lines in cytotoxicity assays. Being aware of the necessity of future efforts to confirm the present findings in a brighter panel of cells and cell lines, these results might provide a first step towards a new understanding of LIR-1.
Available data do not sufficiently support the direct implication of LIR-1 in NK cell inhibition. Upregulation of the receptor does not necessarily favor immunosuppression but might correlate with the acquisition of memory. LIR-1 surface expression increases during B cell and DC maturation [
LIR-1 seems to have a high impact on regulating the balance between activation and inhibition during immune responses [
Within the present study, no alteration of NK mediated cytotoxicity against MM was observed after blockade of LIR-1. Being the only functional inhibitory receptor within this setting, major known side effects by, for example, KIR have been ruled out. This unexpected outcome opens the door to fruitful discussions about the complexity of LIR-1 interactions and its potential role within tumor defense. It is very likely that the present presumptions about functions of LIR-1 within immune regulation are far behind the real impact. We hypothesize that LIR-1 has a key role as a rheostat of NK cell modulation and is strongly involved in the acquisition of NK cell memory.
The authors have no financial conflict of interests.
The authors thank the members of the Tumorimmunology Laboratory for extensive discussions; FACS Core Sorting Unit at University Hospital Hamburg-Eppendorf (UKE) for helpful assistance; D. Niedzielska and U. Kirchner for flexible organization of the Isotope Lab Facility at UKE; Professor Dr. B. Fehse for critical reading of the paper.