Increased expression of non-functional killer ... - Wiley Online Library

British Journal of Haematology 2000, 109, 46±53

Increased expression of non-functional killer inhibitory receptor CD94 in CD81 cells of myeloma patients B A R BAR A B E S O S T R I , 1 E L O I S E B E G G I ATO, 1 A L B E RTO B I AN C H I , 1 S AR A M A R I AN I , 1 M A RTA C OS C I A , 1 S I LV I A P E O L A , 1 M Y R I AM F O G L I E TTA , 1 M A R I O B OC CA D ORO, 1 A L E S S A N D RO P I L E R I , 1 L O R E N Z O M O R E T TA 2 , 3 and M A S S I M O M A S S A I A 1 1 Divisione di Ematologia dell'Universita 0 di Torino, Azienda Ospedaliera San Giovanni Battista di Torino, Torino, 2 Istituto Nazionale per la Ricerca sul Cancro and Centro delle Biotecnologie Avanzate, Genova, and 3 Dipartimento di Medicina Sperimentale, Universita 0 di Genova, Genova, Italy

Received 15 July 1999; accepted for publication 2 December 1999

Summary. Different MHC class I-specific killer inhibitory receptors (KIRs) are expressed in vivo by a minor fraction of activated memory CD81 cells. It has been postulated that KIRs may `fine-tune' specific responses by altering their threshold of activation by the TCR±CD3 complex. We have previously shown that, in multiple myeloma (MM) patients, a large fraction of peripheral blood CD81 cells display the phenotype of chronically activated memory T cells (CD381, HLA-DR1, CD252, CD45R01, CD282). We investigated the expression of KIRs on MM T cells and determined their possible influence on cytolytic responses elicited via the CD3±TCR complex. The expression of CD94, a molecule that is part of a heterodimeric KIR recognizing the nonclassical MHC surface HLA-E molecule, was almost threefold higher in MM T cells than in age-matched normal control subjects (P , 0´0001). CD94 expression was preferentially confined to CD81 cells but not restricted to activated (HLADR1) and/or memory (CD45R01) T cells. Unlike normal T cells, in which CD94 is assembled with glycoproteins of the

NKG2 family to form functional receptors with activating or inhibitory properties, most CD941 MM T cells were devoid of both the NKG2-A and NKG2-C glycoproteins detected in the inhibitory or activating form respectively. CD94 blockade did not significantly affect either T-cell proliferation or cytotoxic T-lymphocyte generation induced by the myeloma-derived cell lines NCI and RPMI 8226. Similarly, the cytolytic activity induced by direct anti-CD3-mediated targeting of MM T cells to FCR1 P815 target cells was unaffected by the addition of anti-CD94 and/or anti-NKG2-A/C monoclonal antibodies (mAbs). These data indicate that the large majority of MM CD81 cells do not express a functional CD94 receptor. Thus, their ability to `fine-tune' an appropriate immune response against tumour cells can be impaired.

INTRODUCTION

some of the mechanisms behind T-cell dysfunction has shown that (1) MM T cells display a dysregulated bcl-2 and Fas expression leading to enhanced susceptibility to spontaneous and induced apoptosis (Massaia et al, 1995) and (2) there are alterations in the molecular organization of the TCR±CD3 complex (Bianchi et al, 1997). These findings, however, provide only a partial explanation for the impaired ability of MM T cells to exert an effective antitumour activity in vivo. A number of data have recently shown that inhibitory receptors can be involved in the impairment of an efficient cytotoxic T lymphocyte (CTL)-mediated anti-tumour response in cancer-bearing hosts (Pardoll, 1996). It has

There is much evidence to show that multiple myeloma (MM) T cells specifically recognize tumour cells (Dianzani et al, 1988; Osterborg et al, 1991; Wen et al, 1998) and that a potent anti-plasma cell activity can be generated in vitro by appropriate stimulation of either peripheral blood or bone marrow T cells (Massaia et al, 1991, 1993). Nevertheless, it is clear that T cells are unable to hold the disease in check, at least in its clinically evident stage. Our investigation of Correspondence: Dr Massimo Massaia, Divisione Universitaria di Ematologia, Via Genova 3, 10126 Torino, Italy. E-mail: maxmass @iol.it q 2000 Blackwell Science Ltd

Keywords: myeloma, inhibitory receptors, T lymphocytes, CD94, cytotoxicity.

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CD94 Overexpression in T cells from Human Myeloma been established that natural killer (NK) cells express MHC class I-specific inhibitory receptors, termed `killer inhibitory receptors'(KIRs). Upon binding to MHC class I molecules on target cells, these receptors deliver a negative signal that prevents the NK-mediated lysis of target cells (Moretta et al, 1996, 1997). Two molecularly distinct KIR families have been identified: (1) the Ig superfamily, including p58´1, p58´2, p70 and p140 molecules, which recognize specific groups of HLA allotypes, and (2) the C-type II lectin superfamily, including the CD94± NKG2-A receptor complex, which operationally senses the overall expression of HLA class I molecules (Moretta et al, 1996, 1997). KIRs have been detected on a subset of human T lymphocytes (usually represented by CD81 cells) in peripheral blood and lymphoid organs (Mingari et al, 1996a,b). Further characterization of KIR1 T cells has revealed that (1) they consistently express a memory phenotype (CD45R01, CD282, CD291, CD18bright), (2) the cells are characterized by a skewed TCRBV repertoire and (3) they are oligoclonal or monoclonal (Mingari et al, 1996b). KIRs may thus be expressed in vivo by T cells exposed to a prolonged antigendriven stimulation (De Maria et al, 1997). Experimental data have shown that their de novo expression can be induced in vitro by stimulation of T cells in the presence of interleukin 15 (IL-15) and transforming growth factor b1 (TGF-b1) (Mingari et al, 1998a; Bertone et al, 1999). Importantly, KIRs may inhibit T-cell activation elicited via the TCR±CD3 pathway, thus impairing T-cell function, including cytolytic activity and cytokine production (Mingari et al, 1996a; De Maria et al, 1997; Mingari et al, 1998a,b; Bertone et al, 1999). We have demonstrated that chronically activated memory T cells (CD381, HLA-DR1, CD25-, CD45R01, CD282) constitute a large fraction of CD81 cells in the peripheral blood of MM patients. They include idiotype-reactive T cells and appear to be negatively correlated with diagnosis and disease progression (Dianzani et al, 1988; OmedeÁ et al, 1990). The aim of this study was to investigate the expression of KIR on MM T cells and to determine their influence on T-cell responses. PATIENTS AND METHODS Patients. Thirty-eight MM patients were studied. All were classified as stage III according to the Durie and Salmon staging system (Durie & Salmon, 1975). Patients receiving treatment were studied at least 3 weeks after the last day of chemotherapy. Patients were not on antibiotics, did not have infections and had not received transfusions for at least 10 d before the study. The control group consisted of 26 agematched normal donors. Antibodies and reagents. The following KIR-specific mAbs were used: EB6 (IgG1, antip58´1), GL183 (IgG1, antip58´2), XA185 (IgG1, anti-CD94), Z27 (IgG1, antip70), Z199 (IgG2b, anti-NKG2-A) and P25 (IgG1, anti-NKG2-A/C). Their production and characterization have already been reported (Moretta et al, 1990; Sivori et al, 1996; Cantoni et al, 1998). Fluorescein-isothiocyanate (FITC)- and phycoerythrin (PE)conjugated anti-isotype goat anti-mouse, FITC-conjugated anti-CD3 (IgG2a), Tri-Colour (TC)-conjugated anti-CD3

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(IgG2a), FITC-conjugated anti-CD4 (IgG2a), FITC-conjugated anti-CD8 (IgG2a), and unconjugated anti-CD14 (IgG2a), anti-CD16 (IgG1), and anti-CD20 (IgG3) mAbs were obtained from Caltag, Burlingame, CA, USA. FITC-conjugated antiHLA-DR (IgG1) was from Becton Dickinson, San Jose, CA, USA. FITC-conjugated anti-CD45R0 (IgG2a) was obtained from Immunotech, Marseille, France. The anti-CD3 mAb used in the cytotoxicity assays was from Cilag, Milan, Italy. Recombinant human IL-2 (18 106 IU/mg specific activity) was from Eurocetus, Milan, Italy. Cells. Peripheral blood mononuclear cells (PBMCs) were obtained by density-gradient centrifugation (Ficoll-Hypaque) of heparinized venous blood. These were then counted microscopically. Their viability, as determined by the trypan blue exclusion dye test, was . 98%. The cells were cultured in RPMI-1640 medium (from Mascia Brunelli, Milan, Italy) containing 10% fetal calf serum (FCS), 2 mmol/l glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco, Milan, Italy). PBMCs were depleted of monocytes by plastic adherence. T cells were then isolated by removing CD141, CD161 and CD201 cells by panning (Dianzani et al, 1988). Briefly, bacteriological-grade Petri dishes were coated overnight with 10 ml of rabbit anti-mouse immunoglobulins (Z109) (from Dako Immunoglobulins, Copenhagen, Denmark) at 10 mg/ml in Tris-HCl 0´05 mol/l, pH 9´5, at 48C. Unbound antibody was removed by washing the dishes three times with phosphate-buffered saline (PBS) 1 5% FCS, and residual binding sites were saturated by a 1-h incubation at 48C with PBS 1 5% FCS. PBMCs were incubated with three mAbs (unconjugated anti-CD14, anti-CD16 and anti-CD20) for 30 min at 48C, washed twice with cold PBS 1 5% FCS, and then incubated for 2 h at 48C in PBS 1 5% FCS in pretreated dishes. Non-adherent cells were collected by decanting and gentle washing with PBS 1 5% FCS. The RPMI 8226, NCI, and P815 cell lines (from the American Type Culture Collection, ATCC, Rockville, MD, USA) were grown in RPMI-1640 medium supplemented with 10% FCS, 2 mmol/l glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Cell lines were found to be mycoplasma free after screening with the Mycoplasma Hoechst stain kit (from ICN Biomedicals, Aurora, OH, USA), and maintained in the logarithmic phase of growth at densities of less than 2 105/ml (NCI, RPMI 8226) or 10 103/ml (P815). Cytofluorometric analyses. Five thousand labelling events were routinely accumulated and analysed for fluorescence on a FACScan (Becton Dickinson). The control sample was used as the reference to divide contour plots into quadrants for the identification of unstained cells (lower left quadrant), cells stained by two mAbs (upper right quadrant) and cells stained by only one mAb (upper left and lower right quadrants). Percentages of positive cells in gated files were referred to the total number of events. T-cell functional assays. Purified T cells were cultured at 1 106/ml in RPMI210% FCS medium in 96-well flatbottomed microtitre plates for 3 d at 378C in a humidified atmosphere of 5% CO2 in air. Soluble IL-2 (Eurocetus) was added at 10 U/ml final concentration. XA185 mAb (CD94) was diluted 1:10 in culture. IL-2-induced proliferation was

q 2000 Blackwell Science Ltd, British Journal of Haematology 109: 46±53

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B. Besostri et al assessed by pulsing cells with 37 kBq of [3H]TdR (1.74 TBq/mmol specific activity from Amersham, Milan, Italy) per well and harvesting 4 h later with a semiautomated sample harvester. The filters were then counted in a liquid scintillation counter and the mean cpm of the triplicate determinations was obtained. Mixed lymphocyte cultures (MLCs) were established in round-bottomed 96-well plates. Purified MM T cells were mixed with irradiated (at 3000 rads) tumour cell lines (1:1), acting as stimulator cells, at a final concentration of 1 106/ml in complete medium with IL-2 at 5 U/ml final concentration. The myeloma-derived cell lines NCI and RPMI 8226 were used as a source of stimulator and target cells. XA185 mAb (CD94) was employed at 1:10 final dilution. MLCs were incubated at 378C in a humidified atmosphere of 5% CO2 in air for 5 d. Cell proliferation was evaluated by pulsing cells with [3H]TdR on day 3 as above. CTL generation was evaluated on day 5 with a standard 4-h 51 Cr-release assay. Briefly, 51Cr-labelled target cells (5 103 cells in 0´1 ml) were mixed in triplicate wells at effector-totarget cell ratios of 20:1, 10:1, 5:1 in round-bottomed microtitre plates. The percentage of specific 51Cr release was calculated from the expression: Experimental-spontaneous 51 Cr released 100 Total releasable-spontaneous 51 Cr released

Fig 1. Expression of KIR in CD31 cells of MM patients and normal controls. Two-colour cytofluorometric analysis of CD3/CD94 coexpression in a normal control (panel A) and in a representative MM patient (panel B). Bar chart representation of the proportions of CD31 cells coexpressing CD94, p70, p58´2 and p58´1. MM bars represent the mean value ^ SE from 38 (CD94) to 21 (p58´1) patients; controls bars are the mean value ^ SE from 26 (CD94) to 14 controls.

where the spontaneous 51Cr released represents the amount of 51Cr released by target cells incubated without effector cells. Total releasable 51Cr is that released by target cells treated with 10 mol/l NaOH. In some experiments, MM T cells were purified by panning and cultured for 5 d in complete medium with IL-2 at 5 U/ml final concentration. The cytotoxicity of the effector cells against FcR-positive P815 target cells was then measured with a standard 4-h 51Cr-release assay in the absence or presence of OKT3 mAb (CD3) (10 mg/ml final dilution), XA185 mAb (CD94) (1:8 final dilution) and P25 mAb (NKG2-A/C) (1:40 final dilution). Briefly, 51 Cr-labelled P815 target cells (5 103 cells in 0´1 ml) were mixed in triplicate wells at effector-to-target cell

Fig 2. Cytofluorometric analysis of CD94/HLA-DR and CD94/CD45R0 coexpression in MM T cells. Freshly isolated PBMC were analysed by three-colour cytofluorometric analysis. Files were gated for CD31 cells (stained with TC-anti-CD3 mAb) and side scatter, and the expression of the indicated antigens is shown as a dot plot. Results are from one representative experiment of 19 (CD94/HLA-DR coexpression) to 6 experiments (CD94/CD45R0 coexpression). q 2000 Blackwell Science Ltd, British Journal of Haematology 109: 46±53

CD94 Overexpression in T cells from Human Myeloma

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Fig 3. Cytofluorometric analysis of CD94/NKG2A and NKG2A/NKG2C coexpression in MM T cells. Freshly isolated PBMC were analysed by three-colour immunofluorescence. Files were gated for CD31 cells (stained with TC-anti-CD3 mAb) and side scatter, and the expression of the indicated antigens is shown as a dot plot. Results are from one representative experiment of 7 (CD94/NKG2-A coexpression) to 3 (NKG2-A/ NKG2-A/C coexpression) experiments.

ratios of 20:1, 10:1, 5:1 in round-bottomed microtitre plates. The percentage of specific 51Cr release was calculated as above. RESULTS Expression of KIR in MM T cells The presence of KIRs was evaluated by two-colour cytofluorometric analysis in T cells isolated from normal donors or MM patients. The mean percentage of CD31CD941 cells was highly increased in MM patients (40´4% ^ 2´9%, n 38) compared with normal donors (14% ^ 5´2%, n 26, P , 0´0001) (Fig 1). By contrast, differences between the mean percentages of T cells expressing the p70, p58´1 and p58´2 molecules were not statistically significant in MM patients (n 21) compared

with the control subjects (n 14) (Fig 1). Longitudinal analyses in selected patients showed that the proportion of CD31CD941 cells was stable, as it did not vary over followup periods of up to 15 months. Correlation between CD94 and HLA-DR/CD45R0 expression A positive linear correlation between the expression of HLA-DR and CD94 molecules was established in MM T cells (r 0´74, P , 0´0001) (data not shown). However, three-colour cytofluorometric analysis showed that CD31 HLA-DR1 cells devoid of CD94 expression and CD31 CD941 cells devoid of HLA-DR expression were detectable in the peripheral blood of MM patients (Fig 2). Similarly, the detection of both CD31 CD45R01 cells devoid of CD94 expression and CD31 CD941 cells devoid of CD45R01 expression indicated that CD94 expression was not

Fig 4. Cytofluorometric analysis of the expression of CD94, NKG2-A and NKG2-C molecules by the CD41 and CD81 subsets in a representative MM patient. MM T cells were purified by panning and then analysed by twocolour cytofluorometric analysis for the coexpression of the indicated antigens. Results are from one representative exeriment of 7. q 2000 Blackwell Science Ltd, British Journal of Haematology 109: 46±53

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B. Besostri et al necessarily restricted to HLA-DR1 or CD45R01 T cells, usually considered as activated and memory cells respectively.

Fig 5. Effect of CD94 masking on the cytolytic activity induced in MM T cells by myeloma-derived cell lines. MM T cells were purified by panning and cultured for 5 d with irradiated RPMI 8226 cells (1:1 ratio) as stimulator cells in complete medium with IL-2 at 5 U/ ml and with or without XA185 mAb (CD94) (1:10 final dilution). After culture, the cytotoxicity of the effector cells against the tumour cells was measured by 51Cr-release assay. Results are expressed as mean cpm ^ SD of 6 experiments, each performed in triplicate. Similar data were observed in 4 experiments with the NCI myeloma-derived cell line.

Fig 6. Effect of CD94 cross-linking on the cytolytic activity induced in MM T cells by mAb-mediated targeting to P815 murine cells. MM T cells were purified by panning and cultured for 5 d in complete medium with IL-2 at 5 U/ml. The cytotoxicity of the effector cells against FcR positive P815 target cells was then measured by 51Crrelease assay in the absence (medium) or presence of OKT3 (CD3), XA185 (CD94) and P25 (NKG2-A/C) mAbs added during the assay. Results are expressed as percentage 51Cr release and refer to the effector:target ratio of 20:1 only. Each bar represents the mean value ^ SD of 6 experiments, each performed in triplicate.

Expression of CD94 and CD94-associated molecules in T-cell subsets The CD94 molecule may covalently assemble with at least two glycoproteins belonging to the NKG2 family to form functional heterodimeric receptors (Lopez-Botet et al, 1997; Pende et al, 1997; Cantoni et al, 1998). The NKG2-A molecule (p43) is specifically recognized by the Z199 mAb, while the P25 mAb recognizes both NKG2-A and NKG2-C (p39). The expression of NKG2-A in MM T cells was analysed. Two-colour cytofluorometric analysis showed that only a small proportion (4 ^ 3%) of CD31 cells coexpressed NKG2-A. A slightly higher proportion (5 ^ 4%) was identified by two-colour staining with the P25 mAb. These data were directly confirmed by three-colour cytofluorometric analysis (Fig 3). Thus, the large majority of CD941 MM T cells do not express either NKG2-A or NKG2-C. Finally, the expression of CD94 and CD94-associated molecules was analysed in CD81 and CD41 subsets by two-colour cytofluorometric analysis of purified T cells. In agreement with previous data obtained in normal donors (Mingari et al, 1996b), their expression was mostly confined to CD81 cells (Fig 4). Remarkably, in some patients the majority of CD81 cells expressed CD94. Analysis of CD94 involvement in the cytolytic activity of MM T cells Initially, the effect of soluble anti-CD94 mAb on IL2-induced T-cell proliferation was investigated. MM and normal T cells were cultured for 3 d in media containing IL-2, either with or without XA185 mAb (CD94). No significant effect was observed in XA185-treated MM and normal T-cell cultures. Next, the effect of soluble XA185 mAb on allogeneic MM T-cell responses was evaluated. MLCs were set up using myeloma cell lines as stimulators and purified MM T cells as responders, in the presence or absence of XA185 mAb. Neither cell proliferation (data not shown) nor the induction of allospecific CTL activity were significantly affected by the mAb (Fig 5). Involvement of CD94 in the cytolytic activity of MM T cells was investigated in a redirected killing assay using FcR1 P815 murine target cells (Bertone et al, 1999). After 5 d incubation in the presence of low doses of IL-2, MM T cells were challenged with P815 cells in the presence or absence of soluble XA185 mAb or P25 mAb (NKG2-A/C). No significant effect on the magnitude of target cell cytolysis was induced by CD94 cross-linking (Fig 6). The assay was also performed in the presence of both OKT3 mAb (CD3) and XA185 mAb or P25 mAb to determine whether CD94 cross-linking regulates the cytolytic activity of MM T cells elicited upon cross-linking of the CD3±TCR complex. Although CD3 cross-linking induced a strong cytolytic activity, P815 cell lysis was not significantly influenced by the simultaneous cross-linking of CD94 or NKG2-A/C molecules, indicating that the largely predominant CD94 complex expressed in MM T cells is devoid of NKG2-A and


CD94 Overexpression in T cells from Human Myeloma NKG2-C and does not substantially enhance or inhibit their cytolytic activity. To rule out the possibility that CD94 down-regulation was responsible for the lack of regulatory activity, MM T cells were stained after the incubation with allogeneic cells and/or IL-2 and found to express unaltered levels of CD94 (data not shown). DISCUSSION This study evaluated the role of KIRs in the immune dysregulation of MM T cells. We focused on these receptors because they are expressed on activated T cells and may negatively regulate cell-mediated immune responses (Mingari et al, 1996a). Determination of the proportion of T cells coexpressing KIRs showed that the expression of CD94 was almost threefold higher in MM T cells than in age-matched normal donors. No differences were observed in the expression of KIRs belonging to the Ig superfamily (p58´1, p58´2, p70). CD94 is surface expressed before any other KIRs in the course of NK-cell maturation and in T cells proliferating in vitro in the presence of IL-15 (Mingari et al, 1998b). CD94 recognizes the non-classical HLA-E molecule, which is only expressed in association with HLA class I alleles sharing a defined signal peptide required for its expression, and therefore operationally senses the overall expression of HLA class I molecules by a potential target cell (Braud et al, 1998; Lee et al, 1998). CD94 was preferentially expressed on MM CD81 cells, as previously reported for normal T cells (Mingari et al, 1996b; Mingari et al, 1998b). However, its expression was not restricted to activated (HLA-DR1) and/or memory (CD45R01) T cells. In individual patients, the proportions of HLA-DR1 and CD941 T cells were positively correlated. Even so, both CD941 HLA-DR- and CD94 HLA-DR1 T-cell subsets were detectable. The same phenotypic heterogeneity was observed with regard to CD45R0 coexpression. These data suggest that the chronic activation state of MM T cells is not the only factor contributing to CD94 expression. Alternatively, the variable expression of CD94, HLA-DR, and CD45R0 may reflect different stages of chronic T-cell activation. It has become clear that CD94 is expressed on the cell surface as a heterodimeric receptor complex consisting of CD94 itself associated with glycoproteins belonging to the NKG2 C-type lectin family (NKG2-A/B, NKG2-C, NKG2-E and NKG2-F) (Sivori et al, 1996; Lopez-Botet et al, 1997; Pende et al, 1997; Cantoni et al, 1998). The NKG2-A gene codes for the p43 subunit responsible for inhibitory signalling and is recognized by the Z199 mAb. We found a very low proportion of NKG2-A1 cells within the CD31CD941 subset, indicating that the large majority of MM T cells do not express the inhibitory form of the CD94 receptor complex. A novel mAb, termed P25, has recently been generated against the NKG2-C molecule, which is part of the activating CD94 receptor complex of NK cells (Cantoni et al, 1998). P25 mAb specifically reacts with both NKG2-C and NKG2-A. When used in combination with the Z199 mAb, it allows discrimination between the

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activating (P25 Z199 ) and inhibitory (P25 Z1991) forms of the CD94 receptor complex. We also found a very low proportion of NKG2-C1 cells, indicating that MM T cells include very few CD941 cells with the inhibitory and/or the activating forms. We have previously observed that the ability of MM T cells to generate alloreactive CTLs and lymphokine-activated killer (LAK) cells is impaired (Massaia et al, 1988, 1990). As this impairment may reflect the expression of inhibitory receptors, we performed a series of functional assays using soluble or cell-bound CD94 mAbs to determine whether CD94 masking or cross-linking had any effect on cytolytic MM T-cell responses. KIR expression has indeed been shown to interfere with the cytolytic T-cell response in both melanoma and patients infected with human immunodeficiency virus (HIV) (De Maria et al, 1997; Ikeda et al, 1997). KIR-mediated inhibitory effects have also been documented in antigen-specific class I-restricted CTL a/b T-cell clones, which are reactive against a melanoma-associated antigen (Ikeda et al, 1997), and g/d T-cell clones, which are reactive against melanoma tumour cells and B-cell lymphoma cell lines (Fisch et al, 1997; Bakker et al, 1998). Finally, CD941/ NKG2-A1 alloreactive CTLs, generated in the presence of IL15, were unable to specifically lyse the relevant allogeneic target cells (Mingari et al, 1998a). CD94 blockade did not significantly affect either T-cell proliferation or CTL generation induced by the myeloma-derived cell lines NCI and RPMI 8226. The cytolytic activity induced by direct antiCD3-mediated targeting of MM T cells to FcR1 P815 target cells was also unaffected by the addition of anti-CD94 and anti-NKG2-A/C mAbs. The use of soluble and cell-bound mAbs thus failed to provide an explanation for the actual function of the CD94 receptor complex expressed in MM T cells. Although CD941 cells lacking both NKG2-A and NKG2-C expression have also been detected in normal individuals, their proportion is usually rather low. In this context, MM T cells represent an unusual finding, and may provide a suitable source of cells to identify novel CD94associated molecules and their method of interference with normal T-cell function. The reason why the large majority of MM T cells express this apparently inactive form of the CD94 receptor complex is unclear. De novo expression of this form may be influenced by certain cytokines, as CD941 T cells generated from normal donors in the presence of IL-15 or TGF-b include a relatively large fraction of cells lacking NKG2-A and NKG2-C as well as the related regulatory function (Mingari et al, 1998a; Bertone et al, 1999). However, non-functional CD94 receptors in normal donors are mostly confined to CD41 cells, rather than CD81 cells as in MM patients. Thus, even if cytokines released by tumour cells may play a role, the chronic activation state of MM T cells, predominantly restricted to CD81 cells, can contribute to the surface expression of non-functional CD94 receptors. The abnormal CD94 expression may explain some of the functional alterations previously described in MM T cells with special regard to their reactivity to CD3 stimulation. Highly purified MM T cells are hyper-reactive to multivalent cross-linking by plastic-immobilized CD3 mAbs, but the very same cells are hyporeactive to phorbol myristate acetate


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(PMA) and ionomycin, phytohaemagglutinin (PHA) and CD2 stimulations (Massaia et al, 1991). MM T cells can secrete large amounts of IL-2 and interferon gamma (IFN-g) and kill autologous plasma cells in vitro (Massaia et al, 1993), but they are also very susceptible to spontaneous and induced apoptosis (Massaia et al, 1995). These abnormalities are mostly confined to chronically activated CD81 cells, i.e. the same cells that are predominantly CD941. Abnormal signalling through the CD3±TCR complex may occur because there are alterations in the molecular organization of the complex itself (Bianchi et al, 1997), but also because there are alterations in regulatory molecules, such as CD94. By lowering or raising the activation threshold of CD3±TCR-mediated responses, the inhibitory or activating CD94 forms can `fine-tune' the activation requirements of T cells, depending on their differentiation stage and activation status. The lack of activating forms may impair the activation of tumour-specific T cells with low-affinity TCR (Mandelboim et al, 1996); on the other hand, the lack of inhibitory forms in activated T cells may facilitate exaggerated signalling such as that observed during multivalent CD3 cross-linking (Massaia et al, 1991). This may not necessarily result in a more effective immune activation, but may rather predispose to activationinduced cell death, an intrinsic feature of chronically activated MM T cells (Massaia et al, 1995). Indeed, we have observed that repeated in vivo infusions of OKT3 and IL-2 in MM patients do not boost T-cell immunity but induce the opposite effect by triggering the apoptosis of activated T cells (Borrione et al, 1996). The finding that a large fraction of CD81 cells do not express a functional CD94 receptor (including naive cells other than memory and activated CD81 cells) suggests that the ability to `fine-tune' an appropriate immune response can be impaired in the effector arm of MM patients. Manipulation of inhibitory receptors has recently been proposed as a novel immunotherapy-based strategy to enhance anti-tumour immunity. In murine cancer models, infusion of mAbs to CTLA-4, a negative regulator of T cell activation, resulted in tumour rejection (Leach et al, 1996). Similarly, it has been proposed that blockade of MHC class I inhibitory receptors may release the brake on KIR1 T cells (and NK and gd T cells), resulting in enhanced anti-tumour activity (Bakker et al, 1998). Our study, which is to date the largest to assess both the phenotypic and the functional profile of CD94 in cancer patients, has shown that this molecule displays some unique features in cancer patients compared with CD94 expressed on normal T cells upon activation. Lastly, the presence of CD81 cells with non-functional CD94 receptors would seem to be a very unique condition of MM patients. There are a few reports about the expression of KIRs on T cells of cancer-bearing hosts, mostly melanoma patients. In these patients, there is a general overexpression of KIRs, including KIRs belonging to the Ig superfamily (Speiser et al, 1999). This is not the case in MM patients, in whom CD94 is the only overexpressed KIR. Moreover, KIRs overexpressed in melanoma patients are clearly inhibitory, since their

blockade and/or ligation enhance tumour-specific cytolytic responses, indicating that the mechanisms by which KIRs impair tumour-specific immune responses are different in MM and melanoma patients. Thus, further studies are required to elucidate the immunoregulatory role of CD94, before its manipulation can be put to profitable use for immune interventions in MM patients. ACKNOWLEDGMENTS We would like to thank Professor John Iliffe for editorial assistance. This work was supported by AIRC (Milan, Italy), MURST 60% (Rome, Italy) and Compagnia San Paolo di Torino (Turin, Italy). Fellowship recipients are: B.B. (Associazione Italiana Amici JoseÂ Carreras, Turin, Italy), S.M. (AIL, Turin, Italy) and S.P. (Comitato Gigi Ghirotti, Turin, Italy). REFERENCES Bakker, A.B., Phillips, J.H., Figdor, C.G. & Lanier, L.L. (1998) Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, gd T cells and antigenspecific CTL. Journal of Immunology, 160, 5239±5245. Bertone, S., Schiavetti, F., Bellomo, R., Vitale, C., Ponte, M., Moretta, L. & Mingari, M.C. (1999) Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. European Journal of Immunology, 29, 23±29. Bianchi, A., Mariani, S., Beggiato, E., Borrione, P., Peola, S., Boccadoro, M., Pileri, A. & Massaia, M. (1997) Distribution of Tcell signalling molecules in human myeloma. British Journal of Haematology, 97, 815±820. Borrione, P., Beggiato, E., Montacchini, L., Pileri, A., Bianchi, A. & Massaia, M. (1996) Clinical and immunological studies in advanced cancer patients sequentially treated with OKT3 and IL-2. Leukemia and Lymphoma, 21, 325±330. Braud, V.M., Allan, D.S., O'Callaghan, C.A., Soderstrom, K., D'Andrea, A., Ogg, G.S., Lazetic, S., Young, N.T., Bell, J.I., Phillips, J.H., Lanier, L.L. & McMichael, A.J. (1998) HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391, 795±799. Cantoni, C., Biassoni, R., Pende, D., Sivori, S., Accame, L., Pareti, L., Semenzato, G., Moretta, L., Moretta, A. & Bottino, C. (1998) The activating form of CD94 receptor complex: CD94 covalently associates with the Kp39 protein that represents the product of the NKG2-C gene. European Journal of Immunology, 28, 327±338. De Maria, A., Ferraris, A., Guastella, M., Pilia, S., Cantoni, C., Polero, L., Mingari, M.C., Bassetti, D., Fauci, A.S. & Moretta, L. (1997) Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: impairment of specific cytolytic functions. Proceedings of the National Academy of Sciences of the USA, 94, 10285±10288. Dianzani, U., Pileri, A., Boccadoro, M., Palumbo, A., Pioppo, P., Bianchi, A., Camponi, A., Battaglio, S. & Massaia, M. (1988) Activated idiotype-reactive cells in suppressor/cytotoxic subpopulations of monoclonal gammopathies: correlation with diagnosis and disease status. Blood, 72, 1064±1068. Durie, B.G.M. & Salmon, S.E. (1975) A clinical staging system for multiple myeloma: correlation of measured myeloma cell mass with presenting features, response to treatment and survival. Cancer, 36, 842±854. Fisch, P., Meuer, E., Pende, D., Rothenfuber, S., Viale, O., Kock, S.,


CD94 Overexpression in T cells from Human Myeloma Ferrone, S., Fradelizi, D., Klein, G., Moretta, L., Rammensee, H.G., Boon, T., Coulie, P.G. & Van der Bruggen, P. (1997) Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human Vg9/Vd2 T cells in tumor immunity. European Journal of Immunology, 27, 3368±3379. Ikeda, H., Lethe, B., Lehmann, F., Van Baren, N., Baurain, J.F., De Smet, C., Chambost, H., Vitale, M., Moretta, A., Boon, T. & Coulie, P.G. (1997) Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity, 6, 199±208. Leach, D.R., Krummel, M.F. & Allison, J.P. (1996) Enhancement of antitumor immunity by CTLA-4 blockade. Science, 271, 1734± 1736. Lee, N., Llano, M., Carretero, M., Ishitani, A., Navarro, F., LopezBotet, M. & Geraghty, D.E. (1998) HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proceedings of the National Academy of Sciences of the USA, 95, 5199±5204. Lopez-Botet, M., Perez-Villar, J.J., Carretero, M., Rodriguez, A., Melero, I., Bellon, T., Llano, M. & Navarro, F. (1997) Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunological Review, 155, 165±174. Mandelboim, O., Davis, D.M., Reyburn, H.T., Vales-Gomez, M., Sheu, E.G., Pazmany, L. & Strominger, J.L. (1996) Enhancement of class II-restricted T cell responses by co-stimulatory NK receptors for class I MHC proteins. Science, 274, 2097±2100. Massaia, M., Dianzani, U., Bianchi, A., Camponi, A., Boccadoro, M. & Pileri, A. (1988) Defective generation of alloreactive cytotoxic T lymphocytes (CTL) in human monoclonal gammopathies. Clinical Experimental Immunology, 73, 214±218 Massaia, M., Bianchi, A., Dianzani, U., Camponi, A., Attisano, C., Boccadoro, M. & Pileri, A. (1990) Defective interleukin-2 induction of lymphokine-activated killer (LAK) activity in peripheral blood T lymphocytes of patients with monoclonal gammopathies. Clinical Experimental Immunology, 79, 100±104. Massaia, M., Bianchi, A., Attisano, C., Peola, S., Redoglia, V., Dianzani, U. & Pileri, A. (1991) Detection of hyperreactive T cells in multiple myeloma by multivalent cross-linking of the CD3/TCR complex. Blood, 78, 1770±1780. Massaia, M., Attisano, C., Peola, S., Montacchini, L., OmedeÁ, P., Corradini, P., Ferrero, D., Boccadoro, M., Bianchi, A. & Pileri, A. (1993) Rapid generation of anti-plasma cell activity in the bone marrow of myeloma patients by CD3-activated T cells. Blood, 82, 1787±1797. Massaia, M., Borrione, P., Attisano, C., Barral, P., Beggiato, E., Montacchini, L., Bianchi, A., Boccadoro, M. & Pileri, A. (1995) Dysregulated Fas and bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients. Blood, 85, 3679±3687. Mingari, M.C., Vitale, C., Schiavetti, F., Cambiaggi, A., Bertone, S., Zunino, A. & Ponte, M. (1996a) HLA-class I-specific inhibitory receptors of NK type on a subset of human T cells. Chemistry Immunology, 64, 135±145. Mingari, M.C., Schiavetti, F., Ponte, M., Vitale, C., Maggi, E., Romagnani, S., Demarest, J., Pantaleo, G., Fauci, A.S. & Moretta, L. (1996b) Human CD81 T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or

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monoclonally expanded cell populations. Proceedings of the National Academy of Sciences of the USA, 93, 12433±12438. Mingari, M.C., Ponte, M., Bertone, S., Schiavetti, F., Vitale, C., Bellomo, R., Moretta, A. & Moretta, L. (1998a) HLA class Ispecific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD81 T cells. Proceedings of the National Academy of Sciences of the USA, 95, 1172±1177. Mingari, M.C., Moretta, A. & Moretta, L. (1998b) Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunology Today, 19, 153±157. Moretta, A., Bottino, C., Pende, D., Tripodi, G., Tambussi, G., Viale, O., Orengo, A., Barbaresi, M., Merli, A., Ciccone, E. & Moretta, L. (1990) Identification of four subsets of human CD3±CD161 natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. Journal of Experimental Medicine, 172, 1589±1598. Moretta, A., Bottino, C., Vitale, M., Pende, D., Biassoni, R., Mingari, M.C. & Moretta, L. (1996) Receptors for HLA-class I-molecules in human natural killer cells. Annual Review of Immunology, 14, 619±648. Moretta, A., Biassoni, R., Bottino, C., Pende, D., Vitale, M., Poggi, A., Mingari, M.C. & Moretta, L. (1997) Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunological Review, 155, 105±117. OmedeÁ, P., Boccadoro, M., Gallone, G., Frieri, R., Battaglio, S., Redoglia, V. & Pileri, A. (1990) Multiple myeloma: increased circulating lymphocytes carrying plasma cell-associated antigens as an indicator of poor survival. Blood, 76, 1375±1379. Osterborg, A., Masucci, M., Bergenbrant, S., Holm, G., Lefvert, A.K. & Mellstedt, H. (1991) Generation of T cell clones binding F(ab 0 )2 fragments of the idiotypic immunoglobulin in patients with monoclonal gammopathy. Cancer Immunological Immunotherapy, 34, 157±162. Pardoll, D. (1996) Releasing the brakes on antitumor immune response. Science, 271, 1691. Pende, D., Sivori, S., Accame, L., Pareti, L., Falco, M., Geraghty, D., Le Bouteiller, P., Moretta, L. & Moretta, A. (1997) HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex. European Journal of Immunology, 27, 1875±1880. Sivori, S., Vitale, M., Bottino, C., Marcenaro, E., Sanseverino, L., Parolini, S., Moretta, L. & Moretta, A. (1996) 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. European Journal of Immunology, 26, 2487±2492. Speiser, D.E., Valmori, D., Rimoldi, D., Pittet, M.J., Lienard, D., Cerundolo, V., MacDonald, H.R., Cerottini, J.C. & Romero, P. (1999) CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. European Journal of Immunology, 29, 1990±1999. Wen, Y.J., Ling, M. & Lim, S.H. (1998) Immunogenicity and crossreactivity with idiotypic IgA of VH CDR3 peptide in multiple myeloma. British Journal of Haematology, 100, 464±468.
