Effect of tumor growth factor-beta on NK receptor expression by allostimulated CD8+ T lymphocytes.

ARTICLE

INTRODUCTION

Experimental evidence has been provided indicating that TGF-ß is the most potent immunosuppressive cytokine that modulates proliferation, differentiation and function of T cells [1]. In this context, we have previously shown that TGF-ß, added at the sensitizing phase of MLR, resulted in the inhibition of the allogeneic cytotoxic and proliferative T cell response [2]. Because TGF-ß has been reported to modulate the expression of receptors important in cell activation and differentiation, we investigated its role in the induction of NK receptors during allostimulation.

The recent identification of the family of NK receptors (NK-R) and the characterization of both inhibitory and activating signals mediated by the different members of the family have lead to a better understanding of the mechanisms regulating target cell recognition and NK and T cell activation [3, 4]. These specific receptors expressed on overlapping NK subsets recognize polymorphic determinants on HLA molecules and their interaction with HLA molecules results in the delivery of a negative signal by NK cells leading to target cell protection [5]. In humans, NK receptors belong to two distincts molecular families: one corresponding to the Ig superfamily such as HLA-C specific p58 [6-8], HLA-B specific p70 [9] and the other including type II membrane proteins, represented by CD94 expressed as an heterodimer associated with NKG2 molecules [10, 11]. The latter receptor displays a broad expression on peripheral NK cells and is involved in the recognition of different HLA-I molecules on target cells [12]. It has been reported that CD94/NKG2 heterodimer serves as a receptor for HLA-E non-classical HLA-I molecules [13]. Membrane expression of HLA-E molecules is dependent on the binding to signal peptides from most HLA-A, B, C and G proteins thus explaining the broad range of CD94 specificity. CD94 is expressed as a heterodimer composed of CD94 disulfide subunit linked to various NKG2 glycoproteins which determine the nature of the transduced signal: NKG2-A, B mediating an inhibitory signal, NKG2-C-D and E an activatory one [14, 15]. Indeed, ligation of CD94 on different NK clones or subsets can both trigger and inhibit cell mediated cytoxicity (16), and induce apoptosis of IL-2 stimulated NK subsets (17). Despite structural differences between both types of NK-R (immunoglobulin and lectin), all these receptors appear to use a common strategy to inhibit or promote NK and T cell activation. Most of both types of receptors contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). Upon receptor engagement with HLA-I molecules, ITIM are tyrosine phosphorylated and recruit protein tyrosine phosphatases that mediate NK cell cytotoxicity inhibition. Some of these receptors do not contain ITIM and can function as activatory receptors [18]. Recent studies have shown that DAP-12 [19], a 12kDa adaptor molecule possessing ITAM can associate with p58 members lacking ITIM and NKG2-C (20) and this interaction results in cellular activation.

Both types of NK-R belonging either to the Ig superfamily or to the lectin family represented by CD94/NKG2 molecules are expressed on peripheral minor CD8⁺ T cell subsets [21]. NK-R engagement leads to inhibition of T cell functions including T cell receptor (TCR)- mediated triggering of cytolytic activity and lymphokine production [22, 23]. The expression of inhibitory receptors that counteract the function of cytotoxic T lymphocytes may have important consequences for the host by modulating the immune response. Therefore it is important to define the mechanisms which determine the expression of NK-R in an immune response. The regulation of NK receptors remains unknown although we have recently shown that IL-15 is a potent inducer of CD94/NKG2-A during differentiation of NK cells from CD34⁺ precursors [24].

In the present study, we show that TGF-ß upregulates NK-receptor expression by allostimulated lymphocytes emphasizing the existence of an additional mechanism used by this cytokine in the control of T cell functions.

MATERIAL AND METHODS

Purification of T cells from PBMC and alloreaction

Blood from normal adult volunteers was obtained from batch leukopheresis (Blood Bank, Saint Louis Hospital, Paris, France). PBMC were isolated by Ficoll/Hypaque density gradient centrifugation and resuspended in RPMI 1640 medium (Life Technologies, Cergy Pontoise, France) supplemented with 10% heat inactivated human serum (J. Boy Institute, Reims France). T cells were further purified by Percoll gradient centrifugation as previously described to eliminate monocytes and NK cells. Purified T cells were incubated for 6 to 10 days with irradiated EBV transformed B cells E418 (HLA A1, B52, DR2) at a ratio of 4 to 1. At the onset of the culture or 48 hours after the beginning of the stimulation, cytokines were added in separate cultures: IL-2 (5 ng/ml), IL-15 (10 ng/ml) and TGF-ß (0.5, 1 and 2.5 ng/ml). Human IL-2 was kindly provided by Roussel Uclaff, Romainville, France. TGF-ß and IL-15 were purchased from R&D systems (Abingdon, Oxon, UK) and from Innotest (Besançon, France) respectively.

Assay for proliferation

Purified T cells (10⁵ cells/well) were plated in round bottom, 96 well plates with irradiated (6,000 rad) stimulating E418 cells (0.25 x 10⁵ cells/well). Triplicate cultures in 0.2 ml of medium were incubated for 6 days at 37° C in 5% CO₂. Plates were pulsed with ³H TdR (2mCi/well, specific activity 57mCi/mmol, Amersham, Buckinghamshire, UK) during the last 18 hours. Cultures were harvested onto filter paper with a Skatron apparatus (Skatron, Lier, Norway) and radioactivity was counted in a beta scintillation counter (LKB Instruments, Orsay France). Results are expressed as net cpm of ³H TdR incorporation and represent the mean of triplicate cultures.

Immunofluorescence analysis

The phenotype of the alloreactive T cells was analyzed by incubating 10⁵ cells with murine mAb recognizing human antigens that were coupled to phycoerythrin (PE) or FITC in 50 µl RPMI/1% FCS for 20 min on ice. The mAb used recognized the following human antigens: UCHT1 (anti-CD3, IgG1, FITC conjugated), B9.11 (anti-CD8, IgG1, FITC conjugated), B1.49 (anti-CD25, IgG2a, FITC conjugated) and were purchased from Immunotech (Marseille, France). For indirect two color fluorescence, 2 x 10⁵ cells were first incubated with the unlabelled mAb HP3B1 (anti-CD94, IgG2a), Z199 (anti-NKG2A, IgG1), EB6 (anti-CD158a, IgG1) directed against p58.1, GL183 (anti-CD158b, IgG1) directed against p58.2, anti-p70 (Z27, IgG1) and anti-p140 (Q66, IgG1) followed by a FITC or PE conjugated goat anti mouse Ig. After a saturation step with mouse Ig, cells were finally incubated with PE coupled mAb (CD8 or CD3). Background levels were measured using isotypic controls. Analysis was done on a FACS-Sort (Becton Dickinson, Pont de Claix, France). When two color analysis was performed, compensation was set up with single stained samples. Low FSC elements were excluded from the analysis by gating them out and 5,000 events were collected and analyzed using Cellquest software (Becton Dickinson, Pont-de-Claix, France).

Assay for cytolytic activity

The cytolytic activity of allostimulated T cells obtained from the different culture conditions were tested against ⁵¹Cr-labeled E418 cells in the presence or absence of anti-CD94 (HP3B1, IgG2a), anti-NKG2-A (Z199, IgG1) or anti-class I (W6/32, IgG2a). Data are expressed as percentage of specific lysis at the indicated effector/target cell ratios. In some experiments, the cytolytic activity of T cell lines was assessed against the P815 mastocytoma mouse cell line in the presence of purified anti-CD3epsilon mAb (10 µg/ml). Briefly, 2 x 10^{3 51}Cr labeled P815 cells coated with anti-CD3 were incubated with serial dilutions of T cell lines (E/T ratio ranging from 20/1 to 2/1). CD3 redirected lysis of labeled P815 cells was modulated by the presence of indicated mAbs, added at the initiation of the assay.

Analysis of NKG2 mRNA transcripts by RT-PCR

Total RNA were extracted from allostimulated T cells according to a modified guanidinium isothiocyanate-phenol-chloroform extraction method (RNABle, Eurobio, Les Ulis, France). cDNA was prepared by the standard method using reverse transcriptase and oligodeoxythymidine primer (Gibco BRL, Life Technologies, Cergy-Pontoise, France). Amplification reactions were performed in a 25 µl mixture containing 2U of Taq Polymerase (Gibco BRL, Life Technologies, Cergy-Pontoise, France), 200 mM dNTP, 0.5 mM of each primer in presence of 1.5 mM Mg²⁺ in Gibco BRL buffer. The conditions for the amplification of CD94, NKG2-A, C, D and E molecules were those previously described [25].

Statistical analysis

The Student's t test was applied to analyze the data and the level of significance was set at a probability of 0.05 to be considered significant. The unpaired t test was used to compare the data obtained before (T0) and after allostimulation (Medium). The paired t test was used to compare expression of NK-R and modulation of cytotoxic activity of T cells by NK-R in different culture conditions in all the experiments.

RESULTS

Effect of TGF-ß on cytolytic activity of allostimulated T cells

Data shown in Figure 1, indicate that addition of TGF-ß along the primary MLR at day 2, 4 and 6 post-stimulation induced a significant decrease in CTL generation. The lytic activity against the stimulator cells E418 in the presence of TGF-ß (2.5 ng/ml) was significantly reduced in all the experiments with a mean decrease of 43% (range 16-90%, n = 9). In contrast, in the same series of experiments, addition of IL-15 did not result in a significant increase of the allostimulated T cell lytic activity.

Effect of TGF-ß on CD94/NKG2-A induction

We wanted to know whether the inhibitory effect of TGF-ß interferes with NK-R expression by allostimulated T cells. Double color fluorescence analysis was therefore performed at the end of the allostimulation to examine the expression of these receptors on CD8⁺ T cells.

Data shown in Figure 2 indicate that on purified resting T cells, CD94 and NKG2-A molecules were expressed on a small subset of CD8⁺ T cells (2.5% and 2.6% respectively). The expression of Ig-like NK receptors (namely p58, p70 and p140) was hardly detected. It is also shown in this figure that following allostimulation, an increased expression of CD94 and NKG2-A was observed as compared to that of resting CD8⁺ T cells, since 9.5% and 10.5% of CD8⁺ T cells expressed CD94 and NKG2-A respectively (Figure 2). Thus, allostimulation in the absence of exogenously added cytokines induced an increased expression of CD94 and NKG2-A on activated T cells: in a series of 12 independent experiments, CD94 expression was detected on 18% of CD8⁺ cells (range: 10-31%) and 16% expressed NKG2-A (range 10.5-24%). CD94⁺ CD8^-/low and NKG2-A⁺ CD8^-/low cells were detected at the end of stimulation. These cells probably correspond to NK cells stimulated by cytokines since CD4⁺ T cells did not express CD94 NKG2-A (data not shown). Most CD94⁺ cells expressed CD25, the IL-2Ralpha chain (data not shown). In addition, no induction of membrane expression of Ig-like receptors could be detected after allostimulation.

In the presence of TGF-ß (2.5 ng/ml), a significant increase of CD94 and NKG2-A expression on T cell blasts was detected as compared to control cultures. As shown in one representative experiment in Figure 2, CD94 and NKG2-A were expressed on 15.5% and 17% of CD8⁺ T cells in the presence of TGF-ß versus 9.5% and 10.5% in medium. Although the percentages of CD94⁺ and/or NKG2A⁺ cells varied from one experiment to another, a paired t test analysis performed on the 12 experiments, showed a significant increased expression of both molecules CD94 (p = 0.02) and NKG2-A (p = 0.01) when TGF-ß was added to the culture. On the other hand, IL-15 did not significantly increase CD94 or NKG2-A expression on T blast cells as compared to cultures in medium (Figure 2) in the same series of alloreactions.

Effect of blockage of the CD94/NKG2-A receptor on the allogeneic cytotoxic response

To examine the role of NK-R on the functional activity of alloreactive T cells, cytotoxicity assays were performed in the presence of specific mAbs to block the interaction between CD94/NKG2-A and their ligands. Blockage of the interaction between CD94/NKG2 and its ligand modulates the lysis differently depending of the amplitude of the cytotoxic response. In medium conditions, addition of anti-NKG2-A or CD94 mAbs during the cytotoxic assay increased the cytolysis of E418 target cells by allostimulated T cells in 3/4 experiments in which lysis was < 30% at an E/T ratio = 15/1 as shown for a representative experiment in Figure 3 (panel A). In the three other experiments in which lysis was high (> 50%, E/T ratio = 10/1), addition of anti-CD94 or NKG2-A mAbs during the effector phase resulted in a slight decrease of lysis (Figure 3, panel B). The same trends in the modulation of lysis were obtained for TGF-ß and IL-15 culture conditions (Figure 3), suggesting that the CD94/NKG2 effect may depend on the activation state of the T cells. To confirm the inhibitory effect of CD94/NKG2-A, CD3-redirected lysis of murine P815 assays were performed. In 3 out of 3 experiments, both NKG2-A and CD94 mAbs significantly inhibited the lysis of CD3 the redirected lysis of P815 by allostimulated the T cells (Figure 4). It noteworthy that in TGF-ß cultures, percentages of inhibition induced by NKG2-A were superior to those observed in other cultures, confirming the increased expression of this receptor in response to TGF-ß.

Expression of NKG2-C, D, E molecules in allostimulated T cells

To explain the dual functionality of the CD94 receptor observed in blockage experiments, expression of different NKG2 molecules which associate with CD94 were analyzed by RT-PCR using specific primers since no mAbs are yet available. NKG2-A transcripts were detected with a higher intensity in allostimulated cells as compared to resting T cells (T0), confirming the results of cytometry. As shown in Figure 5, NKG2 transcripts (NKG2-C, D, E), containing no ITIM but possessing a charged amino acid interacting with DAP12, were present with similar intensities in allostimulated cells from the different culture conditions, suggesting that inhibitory and activatory NKG2 molecules are present in allostimulated T cells.

DISCUSSION

The present study provides evidence that CD8⁺ T lymphocytes activated by alloantigens exhibit an increased expression of the HLA class I specific receptor CD94/NKG2A, as compared to resting CD8⁺ T cells. This expression is probably antigen-dependent since mitogen-driven T cell proliferation in vitro does not induce expression of NK-R [21]. Our data indicate that Ig-like receptors were not induced on T lymphocytes during allostimulation. We further demonstrate an increased expression of CD94 and NKG2-A in the presence of TGF-ß whereas IL-15 had no significant effect.

Concerning the role of IL-15, we have previously shown that this cytokine was essential to the in vitro NK cell differentiation from bone marrow CD34 precursors and that reconstituted NK cells expressed a functional CD94/NKG2-A receptor. However, we cannot exclude that CD94 expression was induced in response to other cytokines since TGF-ß, GM-CSF and TNF-alpha were produced by differentiated NK cells [24]. On the other hand, in a recent study, Mingari et al. [26], using mixed lymphocytes cell cultures from unrelated donors, showed that expression of CD94/NKG2-A was induced only when IL-15 was added during allostimulation. Several differences between the two experimental models may explain this discrepancy. In our study, responder cells correspond to purified T lymphocytes instead of PBMC, avoiding the presence of monocytes and B lymphocytes. The effect of cytokines on PBL could be indirect since cytokines may act on B lymphocytes which in turn may produce cytokines active on T cells [2]. The main difference however, concerns stimulatory cells. EBV transformed B cells used in the present study may express viral antigenic molecules additionally to allogeneic HLA molecules. Such viral molecules may be responsible for the induction of the CD94/NKG2-A receptor on T cells. In this regard, it was reported that CMV-infected cells express molecules that mimic the HLA molecules and protect virus-infected cells from NK cells lysis by ligation of these receptors to the CD94/NKG2 complex [27]. Whether such virus-derived gene products would induce or increase CD94/NKG2 complex expression on NK or T cells in vivo is not known. Furthermore, E418 EBV-transformed B cells may secrete IL-10 and TGF-ß which may explain CD94/NKG2-A induction in medium.

Our data are in agreement with a recent study indicating that TGF-ß-increased expression of CD94/NKG2-A on T cells, stimulated by superantigens [28]. These results reveal a possible additional role of this cytokine in the regulation of an immune response that may have important implications for a better understanding of the immune response directed against tumor cells. Tumor cells, although they may express tumor-specific antigens capable of inducing a CTL response may also secrete immunosuppressive cytokines namely TGF-ß and IL-10. Different histological types such as renal cell carcinoma and non-small lung cell carcinoma produce TGF-ß [29, 30]. Such an environment may favor the expression of NK-R on tumor-infiltrating lymphocytes and thus local production of tumor antigens and TGF-ß may induce proliferation of CD94/NKG2A⁺ T cells. Furthermore, tumor cells may lose expression of HLA class-I molecules down-regulating the activity of the specific CTL resulting in a local immunosuppression [31, 32].

Regarding the effect of CD94/NKG2 on the functional activity of antigen-specific T cells, few data are yet available. Small T cell subset NK-R⁺ T cells in normal donors have been recently shown to be functionally inhibited by recognition of specific HLA-I alleles [22, 33]. Concerning tumor immune response, CD94/NKG2-A⁺ HLA restricted CTL specific for autologous self antigen have been described in melanoma. CTL sharing the same TCR and peptide specificity displayed different expression of inhibitory receptors [34]. Engagement of the CD94/NKG2-A receptor inhibits both antigen-specific cytotoxicity and TNF release of melanoma-specific CTL clones in a peptide dose-dependent fashion [34, 35]. Thus, CD94/NKG2-A inhibitory receptor may modulate the activation threshold of T and NK cells. The dose dependence of TCR ligand for the modulatory role of CD94 described in tumor specific CTL may explain the heterogeneity of our results since it may be assumed that EBV-transformed B cells express large amounts of HLA peptide complexes. Furthermore, bulk T cell populations analysed in the present study may contain several clones expressing inhibitory and activatory CD94/NKG2 receptors as suggested by the results of mRNA analysis of NKG2 proteins.

The present results further outline that Ig type (p58, p70, p140) and lectin type NK receptors may be regulated differently. Although peripheral resting T cells express detectable levels of NK-R belonging to the Ig superfamily, activation by alloantigens in the absence or presence of cytokines do not increase expression of these receptors. Genes encoding the two types of NK-R (Ig type and lectin type) are located on different chromosomes, and thus may be regulated by different mechanisms. Furthermore, it has been recently shown that CD3⁺ p58⁺ T cells from normal donors did not carry TCR specific for alloantigens [36]. Strikingly, emergence of a large donor type CD3⁺CD8⁺ T cell population bearing p58 NK receptor for HLA-C locus alleles was observed in vivo during reconstitution after three loci incompatible (T cell depleted) marrow grafting for acute leukemia [37]. Expansion of p58⁺CD3⁺ T cells was not observed after allogeneic matched bone marrow grafting suggesting that the degree of disparity between donor and host MHC are responsible for such cell expansion. It further suggests that in vitro systems may not completely reflect the in vivo situation for induction and regulation of Ig type and possibly lectin type NK receptors.

Based on the present data, it would be interesting to study the expression of both types of NK receptors to determine if their expression by tumor specific T cells, in response to autologous antigens and locally produced TGF-ß, plays a role in the conflict between the tumor and the immune system of the host [38].

CONCLUSION

Acknowledgements.

This work was supported by grants awarded by INSERM, the Association for Cancer Research ARC (2038 to AC). We would like to thank Yann Lécluse for immunofluorescence analysis and Dr. Alessandro Moretta for providing anti NK-R mAbs: anti CD94, anti NKG2A, anti-p70 and anti-p140.

REFERENCES

1. Moses H L, Yang E Y, Pietenpol J A. 1990. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights (Review). Cell 63: 245.

2. Pardoux C, Asselin-Paturel C, Chehimi J, Gay F, Mami-Chouaib F, Chouaib S. 1997. Functional interaction between transforming growth factor beta and IL-12 in human primary allogeneic cytotoxicity and proliferative response. J Immunol 158: 136.

3. 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. Eur J Immunol 27: 1875.

4. Ryan JC, Seaman WE. 1997. Divergent functions of lectin-like receptors on NK cells. (Review) Immunol. Rev. 155: 79.

5. Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, Moretta L. 1996. Receptors for HLA class-I molecules in human natural killer cells. (Review) Annu. Rev. Immunol. 14: 619.

6. Moretta A, Bottino C, Pende D, Tripodi G, Tambussi G, Viale O, Orengo A, Barbaresi M, Merli A, Ciccone E, et al. 1990. Identification of four subsets of human CD3^­CD16⁺ 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. J. Exp. Med. 172: 1589.

7. Ciccone E, Pende D, Viale O, Than A, Di Donato C, Orengo AM, Biassoni R, Verdiani S, Amoroso A, Moretta A, et al. 1992. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2). J. Exp. Med. 176: 963.

8. Moretta A, Vitale M, Bottino C, Orengo AM, Morelli L, Augugliaro R, Barbaresi M, Ciccone E, Moretta L. 1993. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J. Exp. Med. 178: 597.

9. Litwin V, Gumperz J, Parham P, Phillips J H, Lanier L L. 1994. NKB1: a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J. Exp. Med. 180: 537.

10. Aramburu J, Balboa M A, Ramirez A, Silva A, Acevedo A, Sanchez-Madrid F, De Landazuri M O, Lopez-Botet M. 1990. A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta⁺ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody. J. Immunol. 144: 3238.

11. Chang C, Rodriguez A, Carretero M, Lopez-Botet M, Phillips J H, Lanier L L. 1995. Molecular characterization of human CD94: a type II membrane glycoprotein related to the C-type lectin superfamily. Eur. J. Immunol. 25: 2433.

12. 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. Eur. J. Immunol. 26: 2487.

13. 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.

14. Carretero M, Cantoni C, Bellon T, Bottino C, Biassoni R, Rodriguez A, Perez-Villar J J, Moretta L, Moretta A, Lopez-Botet M. 1997. The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules. Eur. J. Immunol. 27: 563.

15. Houchins J P, Lanier L L, Niemi E C, Phillips J H, Ryan J C. 1997. Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2-C. J. Immunol. 158: 3603.

16. Perez-Villar J J, Melero I, Rodriguez A, Carretero M, Aramburu J, Sivori S, Orengo A M, Moretta A, Lopez-Botet M. 1995. Functional ambivalence of the Kp43 (CD94) NK cell-associated surface antigen. J. Immunol. 154: 5779.

17. Ida H, Robertson M J, Voss S, Ritz J, Anderson P. 1997. CD94 ligation induces apoptosis in a subset of IL-2-stimulated NK cells. J. Immunol. 159: 2154.

18. Vély F, Vivier E. 1997. Conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts. (Review) J. Immunol. 159: 2075.

19. Lanier L L, Corliss B C, Wu J, Leong C, Phillips J H. 1998. Immunoreceptor DAP-12 bearing a tyrosine based activation motif is involved in activating NK cells. Nature 391: 703.

20. Lanier L L, Corliss B, Wu J, Phillips J H. 1998. Association of DAP-12 with activating CD94/NKG2C NK cell receptors. Immunity 8: 693.

21. Mingari M C, Schiavetti F, Ponte M, Vitale C, Maggi E, Romagnani S, Demarest J, Pantaleo G, Fauci A S, Moretta L. 1996. Human CD8⁺ T lymphocytes subsets that express HLA class I specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc. Natl. Acad. Sci. USA 93: 12433.

22. Mingari M C, Vitale C, Cambiaggi A, Schiavetti F, Melioli G, Ferrini S, Poggi A. 1995. Cytolytic T lymphocytes displaying natural killer (NK)-like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production. Int. Immunol. 7: 697.

23. D'Andrea A, Chang C, Phillips J H, Lanier L L. 1996. Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J. Exp. Med. 184: 789.

24. Carayol G, Robin C, Bourhis J H, Bennaceur-Griscelli A, Chouaib S, Coulombel L, Caignard A. 1998. NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions. Eur. J. Immunol. 28: 1991.

25. Valiante N M, Uhrberg M, Shilling H G, Lienert-Weidenbach K, Arnett K L, D'Andrea A, Phillips J H, Lanier L L, Parham P. 1997. Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7: 739.

26. Mingari M C, Ponte M, Bertone S, Schiavetti F, Vitale C, Bellomo R, Moretta A, Moretta L. 1998. HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15 induced expression of CD94/NKG2A in superantigen or alloantigen CD8⁺T cells. Proc. Natl. Acad. Sci. USA 95: 1172.

27. Reyburn H T, Mandelboim O, Vales-Gomez M, Davis D M, Pazmany L, Strominger J L. 1997. The class I MHC homologue of human cytomegalovirus inhibits attack by natural killer cells. Nature 386: 514.

28. Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari M C. 1999. Transforming growth factor-beta-induced expression of CD94/NKG2-A inhibitory receptors in human T lymphocytes. Eur. J. Immunol. 29: 23.

29. Asselin-Paturel C, Echchakir H, Carayol G, Gay F, Opolon P, Grunenwald D, Chouaib S, Mami-Chouaib F. 1998. Quantitative analysis of Th1, Th2 and TGF-beta1 cytokine expression in tumor, TIL and PBL of non-small cell lung cancer patients. Int. J. Cancer 77: 7.

30. Olive C, Cheung C, Nicol D, Falk M C. 1998. Expression of cytokine mRNA transcripts in renal cell carcinoma. Immunol. Cell. Biol. 76: 357.

31. Rivoltini L, Barracchini K C, Viggiano V, Kawakami Y, Smith A, Mixon A, Restifo N P, Topalian S L, Simonis T B, Rosenberg S A, et al. 1995. Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes. Cancer Res. 55: 3149.

32. Geertsen R C, Hofbauer G F, Yue F Y, Manolio S, Burg G, Dummer R. 1998. Higher frequency of selective losses of HLA-A and -B allospecificities in metastasis than in primary melanoma lesions. J. Invest. Dermatol. 111: 497.

33. Ferrini S, Cambiaggi A, Meazza R, Sforzini S, Marciano S, Mingari M C, Moretta L. 1994. T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function. Eur. J. Immunol. 24: 2294.

34. Noppen C, Schaefer C, Zajac P, Schutz A, Kocher T, Kloth J, Heberer M, Colonna M, De Libero G, Spagnoli GC. 1998. C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity. Eur. J. Immunol. 28: 1134.

35. Le Drean E, Vely F, Olcese L, Cambiaggi A, Guia S, Krystal G, Gervois N, Moretta A, Jotereau F, Vivier E. 1998. Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases. Eur. J. Immunol. 28: 264.

36. Yamada N, Nagatami T, Takiguchi M. 1998. T cells expressing killer cell inhibitory receptors do not carry TCR for alloantigens. Hum. Immunol. 59: 488.

37. Albi N, Ruggeri L, Aversa F, Merigiola C, Tosti A, Tognellini R, Grossi C E, Martelli M F, Velardi A. 1996. Natural killer (NK)-cell function and antileukemic activity of a large population of CD3⁺/CD8⁺ T cells expressing NK receptors for major histocompatibility complex class I after "three-loci" -incompatible bone marrow transplantation. Blood 87: 3993.

38. Chouaib S, Asselin-Paturel C, Mami-Chouaib F, Caignard A, Blay J Y. 1997. The host-tumor immune conflict: from immunosuppression to resistance and destruction. (Review) Immunol Today 18: 493.