Natural killer cell-mediated recognition of human trophoblast

seminars in C A N C E R B I OL OG Y, Vol 9, 1999: pp. 13᎐18 Article No. scbi.1998.0108, available online at http:rrwww.idealibrary.com on

Natural killer cell-mediated recognition of human trophoblast Roberto Biassoni†, Cristina Bottino†, Romano MilloU , Lorenzo Moretta†§ and Alessandro Moretta U

Large numbers of natural killer cells are present within the maternal decidua close to the extravillous trophoblast cells. Most of these natural killer cells express high levels of inhibitory NK receptors (KIR) specific for HLA class I molecules. Since trophoblast cells only express HLA-C and HLA-G, the KIR expressed by decidual NK cells can only recognize these HLA class I molecules in order to avoid NK-mediated rejection of fetal tissues. We show that HLA-C recognition by decidual NK cells can be mediated by p58, LIR-1 and (indirectly) by CD94r NKG2A receptors. On the other hand, HLA-G recognition is not only mediated by LIR-1 and (indirectly) by CD94r NKG2A but also by a newly identified receptor termed p49. The p49 receptor, unlike the other KIR, appears to be selectively expressed by decidual NK cells.

been found particularly abundant around infiltrating trophoblast cells suggesting a possible role in regulating the degree of invasion and differentiation of the trophoblast.4 However, the different factors that are involved in the migration and differentiation of NK cells in the uterus are still largely unknown. In this context, NK cells appear to be under the control of hormones since their number increases in the luteal phase when implantation occurs. Moreover, these cells are also increased during estrus and post-ovulatory luteal phase.2 The population of NK cells in the decidual tissues differs from those in circulation with respect to the surface phenotype since they are CD56 bright , CD16y.1 Moreover, the percentage of NK cells expressing a particular HLA class I-specific inhibitory receptor ŽKIR. are increased as compared to circulating NK cells from the same individual. This is particularly true for the p58.1 and p58.2 ŽCD158a and CD158b. HLA-C-specific inhibitory receptors.5 It is of note that invading extravillous trophoblast cells in humans lack the expression of both HLA-A and HLA-B while they express HLA-C and non-classical HLA class I molecules such as HLA-G 6,7 and possibly HLA-E. While the expression of HLA-C molecules by trophoblast cells and the large fraction of decidual NK cells bearing p58 receptors suggests a critical role of this interaction in the protection of trophoblast cells from NK-mediated cytotoxicity the role of HLA-G molecules is still under investigation. Importantly the HLA-G molecules are confined to extravillous trophoblast cells suggesting a specific role of these molecules in protecting trophoblast cells from the maternal immune system.8 HLA-G, uniquely among the HLA class I molecules, display many different isoforms, generated by alternative splicing, and a limited polymorphism that do not affect the residues that are involved in peptide binding.2,9 ᎐ 11 The use as target cells of transfectants expressing

Key words: HLA r ITIM r ITAM r KIR r NK 䊚1999 Academic Press

Introduction PREGNANCY THAT IS CHARACTERIZED by the exposure of maternal cells to fetal tissues is the only occasion in which natural recognition of allogeneic cells occurs. Importantly, large numbers of maternal NK cells, exceeding in number T cells, infiltrate decidual tissues;1,2 in particular, NK cells are present in the early phase of gestation when placental trophoblast cells invade into decidua.3 Indeed, NK cells have U

From the Dipartimento di Medicina Sperimentale, Sezione di Istologia, Universita ` di Genova, Genova, Italy, †Istituto Nazionale per la Ricerca sul Cancro and Centro Biotecnologie Avanzate, Genova, Italy and §Dipartimento di Oncologia Clinica e Sperimentale, Universita ` di Genova, Genova, Italy 䊚1999 Academic Press 1044-579Xr 99r 010013q 06 $30.00r 0

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r NKG2 heterodimers in Involvement of CD94r HLA-G recognition

the HLA-G1 molecules has indicated that at least a fraction of peripheral blood NK cells is equipped with inhibitory receptors that recognize this molecule. However, since peripheral blood NK cells and decidua infiltrating NK cells may display different phenotypic features and distinct responses to regulating factors Žhormones., it is possible that additional mechanisms may control the cytotoxicity mediated by decidual NK cells.

A second family of HLA class I-specific receptors is represented by heterodimers formed by the association of CD94 with different members of the NKG2 family. Thus it has been demonstrated that the receptor formed by the association of CD94 with the NKG2A molecules can inhibit NK cell-mediated lysis.23,24 On the contrary, the CD94rNKG2C heterodimer constituted a triggering receptor able to induce NK cytotoxicity.25 Both the inhibitory and the activating CD94rNKG2 heterodimers have been implicated in the recognition of multiple HLA class I alleles. Importantly, the CD94rNKG2A receptor that is expressed on a variable portion of peripheral NK cells, was found to be expressed on virtually all decidual NK cells.26 Experiments using as target cells HLA- Gq cell transfectants suggested that the CD94rNKG2A heterodimer could recognize HLA-G molecules as well.20 ᎐ 22 The same conclusion was observed also for the CD94rNKG2C triggering receptor.20 More recently, data regarding the specificity of the CD94rNKG2A receptor for HLA class I molecules, have indicated that this heterodimeric receptor is able to recognize HLA class Ib molecules rather than classical HLA class I molecules.27 ᎐ 30 In particular, it appears that this receptor recognizes HLA-E molecules both in soluble form and when expressed at the cell surface. It has been demonstrated that HLA-E expression on the cell membrane can take place only in the presence of a particular set of peptides. These are derived from the leader sequence of various HLA class I molecules that stabilize the conformation of HLA-E as a trimeric complex at the cell surface. Interestingly, the HLA class I-derived leader sequences that are able to stabilize HLA-E are those found in most HLA class I molecules including HLA-C and HLA-G. Consequently HLA-E expression will be possible only when classical or non-classical HLA class I molecules are co-expressed at the cell surface. These findings, while demonstrating the fine specificity of the CD94rNKG2 receptors also provide an explanation for previous data indicating that different HLA class I alleles were capable of protecting target cells from NK- mediated cytotoxicity by CD94rNKG2Aq NK cells. Therefore, operationally, CD94rNKG2Aq cells can sense different HLA class I molecules, including HLA-G1. It is likely that the high proportion of CD94rNKG2Aq cells in decidual NK cells reflects the requirement for recognition of

p58, p70 and p140 receptors for HLA class I alleles do not recognize HLA-G The p58, p70 and p140 inhibitory receptors ŽKIR. are specific for different groups of HLA class I alleles.12,13 Thus p58.1 and p58.2 receptors recognize distinct, non-overlapping subgroups of HLA-C alleles,14 whereas the p70 and the p140 receptors are specific for HLA-BW4 alleles15,16 or for HLA-A3 and A11 alleles, respectively.17 Since some of these KIR have been detected in a high proportion of decidual NK cells, it was possible that, in addition to their specific HLA class I ligands, they could also recognize HLA-G molecules. Indeed, preliminary studies suggested that some KIR might also recognize HLA-G molecules in cell transfectants: in particular, the first study indicated that HLA-G was recognized by both the p58.1 and p58.2 receptors18 whereas, in a second study, this ability was assigned to the p70 receptor.19 It has to be noted, however, that normally only a relatively small subset of peripheral or decidual NK cells express these receptors. As a consequence most NK cells would not be equipped with inhibitory receptors for HLA-G. At variance with the above results subsequent studies from three different labs 20 ᎐ 22 reached the conclusion that p58, p70 and p140 do not recognize HLA-G. On the other hand, as mentioned above, the p58.1 and p58.2 receptors have been shown to be expressed at higher levels in decidual NK cells.5 This is likely to be important for the recognition of HLA-C molecules that are expressed by trophoblast cells. Thus, although unable to recognize HLA-G, the HLA-C-specific p58 receptors may indeed be important for the control of maternal NK cells. Notably, no increments in the expression of p70 and p140 receptors have been detected in decidual NK cells; this would be in line with the lack of HLA-B and HLA-A expression on trophoblast. 14

NK cells and trophoblast

trophoblast cells in which the expression of HLA-C and HLA-G allows HLA-E expression.

sion, normal NK clones appear to represent a more reliable source of effector cells to determine the specificity of a given receptor. Moreover, this allow the analysis of phenotypically distinct effector cells that may or may not co-express other HLA class I-specific NK receptors. Thus, similar to CD94rNKG2A Žsee above., the LIR-1rILT-2 receptor that can be expressed on the majority of decidual NK cells 26 is likely to control the cytotoxicity mediated by maternal NK cells against the HLA-CqrHLA-Gq trophoblast.

LIR-1-dependent HLA-G recognition The LIR-1rILT-2 molecule31,32 belongs to the Ig superfamily and is expressed on variable proportion of monocytes and lymphocytes including B, T and NK cells. The cytoplasmic tail of this molecule contains typical Immunoreceptor Tyrosine-based Inhibitory Motifs ŽITIM. which upon tyrosine phosphorylation associate with the tyrosine phosphatase SHP-1, 31 a member of the inhibitory signal cascade.33 This suggests that LIR-1 may function as an inhibitory receptor in different cell types. Importantly, LIR-1 was found to function as receptor for the cytomegalovirus ŽCMV. UL18 protein.31,34 This implies that UL18 may negatively signal different ŽLIR1q. cell types involved in the immune response. Recently, we demonstrated that LIR-1 molecule expressed by NK cell clones is functioning as a HLAclass I-specific inhibitory receptor.34 Remarkably, different from the other known KIR belonging to the IgSF, LIR-1 displays a broad specificity for different HLA-class I molecules including HLA-G. Thus the specificity of LIR-1 is reminiscent of that described for CD94rNKG2A since both receptors are characterized by the ability to recognize target cells transfected with different HLA class I molecules. However, LIR-1, unlike CD94rNKG2A, also recognizes cells transfected with HLA-Bw4 alleles.34 These alleles are known to have a leader peptide sequence that does not allow surface expression of HLA-E molecules. This implies that LIR-1 molecules directly recognize HLA-Bw4 molecules. Similar results have been obtained by Colonna et al 32 who analyzed the specificity of the LIR-1 receptor by using as effector cells, the NK-L cell line Žinstead of NK clones.. Differently from the above study, however, we find that LIR-1 may also recognize HLA-C. This apparently conflicting result may be explained by the fact that the inhibitory effect of HLA-C alleles is usually detectable in NKG2A-negative NK clones34 and may not be revealed by the analysis of the NK-L cell line that express the NKG2Aq phenotype. Moreover, the use of the NK-L cell line as a source of effector cells appears highly inappropriate since these cells display a rather inefficient cytolytic activity. This is consequent to abnormalities in the expression of triggering receptors involved in the induction of natural cytotoxicity ŽA. Moretta, personal observation.. In conclu-

The p49 molecule is a novel HLA-G-specific inhibitory receptor selectively expressed by decidual NK cells The p49 molecule is a 49-kDa type I transmembrane protein characterized by two Ig-like domains and a 115 amino acid cytoplasmic tail containing a single ITIM.35 The p49 encoding gene is localized on human chromosome 19. We recently showed that p49, similar to other KIR belonging to the Ig-SF, is capable of recognizing allelic forms of HLA-class I molecules, although with a broader specificity. Indeed, soluble chimeric p49 protein specifically binds to some HLA class I alleles including HLA-A3, -B46 and B7 alleles, but not to others Že.g. HLA-A2, Cw3, -Cw4 and -B51 alleles.. Importantly, the p49 receptor specifically binds to HLA-G1 suggesting its possible role in the control of motherrfetus interactions.26,35 The generation of a p49-specific antiserum allowed us to analyze the surface expression of this novel receptor on lymphocytes derived from pregnant and non-pregnant women. We first analyzed the reactivity of the p49-specific antiserum with peripheral blood lymphocytes isolated from control non-pregnant and from women during the first trimester of pregnancy. While a fraction of lymphocytes expressed NKG2A andror LIR-1 receptors, no staining with anti-p49 antiserum could be detected.26 We further investigated whether p49 was expressed by NK cells isolated from maternal decidua during the first trimester of pregnancy. In this case NK cells represented approximately 70᎐75% of the total mononuclear cell population. In agreement with previous data,5 a substantial fraction of these NK cells were CD56 bright CD16y. In addition, they expressed CD94rNKG2A and LIR-1 receptors. Perhaps more importantly, a fraction of NK cells expressed p49.26 These data suggest that p49 may be selectively expressed by NK cells associated to maternal decidua. 15

R. Biassoni et al

Are trophoblast cells recognized by nonHLA-specific triggering NK receptors?

Thus, these NK cells not only express CD94rNKG2A and LIR-1, but are also equipped with an additional HLA-G1-specific receptor which appears to be absent in peripheral NK cells. Additional information on the various receptors which may sense the HLA-G1 expression in trophoblastic cells, were obtained by the analysis of mononuclear cells isolated from samples of placenta at term.26 In this case, approximately half of these cells displayed the CD56q phenotype. Notably, different from NK cells isolated from maternal decidua during the first trimester, most CD56q cells were also CD16q. Moreover, the majority of NK cells expressed CD94rNKG2A and virtually all were LIR-1q. Analysis of these NK cells with the anti-p49 antiserum demonstrated that virtually all CD16q cells expressed p49. On the other hand, anti-p49 antiserum did not stain CD3q cells present in the same cell suspension. These data indicate that most NK cells isolated from decidua co-express different HLA-G1-specific receptors able to recognize trophoblastic cells. Indeed, the analysis of freshly-isolated decidual NK cells, isolated from placenta at delivery, showed that these NK cells are unable to lyse 221-G1 transfectants. On the other hand, restoration of lysis could be detected in the presence of anti-CD94 or anti-LIR-1 mAb. Remarkably, maximal restoration occurred in the presence of anti-p49 antiserum.26 Experiments of RT-PCR performed on RNA isolated from NK cell clones demonstrated that the p49-specific transcript is expressed in all peripheral blood NK cells in spite of their p49y surface phenotype.35 Thus, p49 surface expression may be acquired by NK cells in the decidual microenvironment, possibly as a consequence of their response to a particular set of cytokineŽs. andror hormoneŽs.. Along this line, p49 may be down-regulated once NK cells leave the placenta, as suggested by the finding that no detectable surface p49 reactivity could be demonstrated in peripheral NK cells from pregnant women. One may speculate that p49 is preformed by NK cells and stored in the cytoplasm. This would allow a rapid surface expression that could prevent the attack of trophoblastic cells by NK cells trafficking to maternal decidua. Altogether these data indicate that the HLA-G1specific p49 receptor is expressed by NK cells in placenta and may cooperate with other receptors to down-regulate NK cell cytotoxicity upon binding to HLA-G1 molecules expressed on placental trophoblastic cells.

The complex mechanisms that are involved in the regulation of the cytotoxicity mediated by decidual NK cells may not simply be based on the interaction of HLA-GrHLA-C-specific inhibitory receptors with their ligands. For example, other non-inhibitory mechanisms may be involved that contribute to the regulation of decidual NK cell function. In this context, the natural cytotoxicity of decidual NK cells against HLA-negative target cells appears to be significantly reduced as compared to that of peripheral blood NK cells.26 This may suggest that in the decidua the mechanisms leading to triggering of natural cytotoxicity Ži.e. the interaction between triggering receptors and their non-HLA ligands. may be at least partially impaired. So far the nature and the specificity of these nonHLA-specific triggering receptors have remained elusive. Several surface molecules which can mediate NK cell triggering have been identified, however, their actual role in natural cytotoxicity still has to be clarified. The low affinity receptor for the Fc portion of IgG ŽFc␥ RIII or CD16. is expressed on most peripheral NK cells but is not expressed by decidual NK cells during early pregnancy.2,26 Since CD16 is a potent inducer of NK cell triggering this may suggest that the low natural cytotoxicity may reflect the low degree of CD16 expression. On the other hand, CD16 is expressed by NK cells derived from placenta at term.26 Such CD16q NK cells are still poorly cytolytic against HLA-negative target cells. These data, together with the fact that there is no evidence so far that CD16 may mediate functions other then ADCC against IgG-coated target cells, appears to rule out a possible role of CD16 as a triggering receptor involved in natural cytotoxicity mediated by decidual NK cells. Recently we have focused our studies on novel putative NK receptor structures characterized by the ability to trigger natural cytotoxicity. The search for these receptors structures led us to the identification of two novel triggering receptors termed NKp46 36,37 and NKp44.38 The NKp46 receptor is strictly confined to both resting and activated NK cells. The unique cellular distribution and the function of the NKp46 receptor suggest that it may be a candidate for a triggering receptor involved in the recognition of non-HLA ligands expressed on NK susceptible targets.36 In line with this hypothesis, NKp46 masking by the specific 16

NK cells and trophoblast

mAb, could significantly inhibit the cytolytic activity of NK cell clones against NK susceptible target cells. Cross-linking of NKp46 molecules not only trigger cytotoxicity but also induce wCa2q x i increases and lymphokine production.36 The NKp46 receptor is a type I transmembrane glycoprotein belonging to the lg superfamily encoded on human chromosome 19 and characterized, in the extracellular portion, by two Ig-like domains of the C2-type. 37 The transmembrane region contains a charged amino acid ŽArg. and the short cytoplasmic tail lacks Immunoreceptor Tyrosine-based Activating Motifs ŽITAM.. Thus NKp46 seems to require the association with ITAMcontaining molecules that allow the transduction of the signalling events that result in triggering of NKmediated cytotoxicity. The CD3␨ and Fc␥ RI ITAMcontaining polypeptides were shown to be associated with NKp46.38 A possible view of the mechanisms of NK cell triggering against HLA-negative target cells is that multiple receptors may co-operate to induce optimal NK cell activation. This does not imply that all triggering receptors function simultaneously since their function may depend upon the presence andror the density of their specific ligands on target cells. Thus NKp46 may not be the ‘unique’ activating receptor and it may co-operate with other ‘triggering receptors’ to induce optimal NK cell activation. Along this line, we showed that another triggering receptor, termed NKp44,38 may co-operate with NKp46 in the induction of natural cytotoxicity. The NKp44 receptor, unlike NKp46, is selectively expressed by activated NK cells while lacking on resting NK cells. Signal transduction via NKp44 seems to require the association with the recently characterized KARAPrDAP12 subunits.38 The low levels of natural cytotoxicity mediated by decidual NK cells may be the consequence of an insufficient cross-linking of the above triggering receptors. This, in turn, may be due to the lack of expression of the receptors themselves or of their specific ligands on trophoblast cells. Along this line, we have recently observed that NK cells derived from peripheral blood of distinct donors express different levels of surface NKp46 molecules. At the clonal level, the natural cytotoxicity of NK cells clearly correlates with the surface density of the NKp46 receptor. Thus it will be crucial to determine whether decidual NK cells express triggering receptors such as NKp46 and NKp44 and whether their surface density is comparable to that of peripheral blood NK cells. Furthermore, as mentioned above, the optimal func-

tion of the triggering receptors may depend upon the surface density of their non-HLA ligands expressed on target cells; the molecular identification of such ligands will allow the analysis of their distribution on NK-susceptible target cells as compared to trophoblast cells.

Acknowledgements This work was supported by grants awarded by the Associazione Italiana per la Ricerca sul Cancro ŽA.I.R.C.., Istituto Superiore di Sanita ` ŽI.S.S.., Consiglio Nazionale delle Ricerche ŽP.F. Biotecnologie., and Ministero dell’Universita ` e della Ricerca Scientifica e Tecnologica ŽM.U.R.S.T..

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15. Litwin V, Gumperz JE, Parham P, Phillips JH, Lanier LL Ž1994. NKB1: a Natural Killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J Exp Med 180:537᎐543 16. Vitale M, Sivori S, Pende D, Augugliaro R, Di Donato C, Amoroso A, Malnati M, Bottino C, Moretta L, Moretta A Ž1996. Physical and functional independency of p70 and p58 NK cell receptors for HLA-class I. Their role in the definition of different groups of alloreactive NK cell clones. Proc Natl Acad Sci USA 93:1453᎐1457 17. Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, Di Donato C, Accame L, Bottino C, Moretta A, Moretta L Ž1996. The Natural Killer cell receptor specific for HLA.A allotypes: a novel member of the p58rp70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140 kD disulphide-linked dimer. J Exp Med 184:505᎐518 18. Pazmany L, Mandelboim O, Vales-Gomez M, Davis DM, Rey´ burn HT, Strominger JL Ž1996. Protection from natural killer cells-mediated lysis by HLA-G expression on target cells. Science 274:792 19. Munz C, Holmes N, King A, Loke YW, Colonna M, Schild H, Rammensee HG Ž1997. Human histocompatibility leukocyte antigen ŽHLA-G . molecules inhibit NKAT3 expressing natural killer cells. J Exp Med 185:385 20. Pende D, Sivori S, Accame L, Pareti L, Falco M, Geraghty D, Le Bouteiller P, Moretta L, Moretta A Ž1997. HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex. Eur J Immunol 27:1875 21. Perez-Villar JJ, Melero I, Navarro F, Carretero M, Bellon T, Llano M, Colonna M, Geraghty DE, Lopez-Botet M Ž1997. The CD94rNKG2A inhibitory receptor complex is involved in the natural killer cell-mediated recognition of cells expressing HLA-G1. J Immunol 158:5736 22. Soderstrom K, Corliss B, Lanier LL, Phillips JH Ž1997. ¨ CD94rNKG2 is the predominant inhibitory receptor involved in recognition of HLA-G by decidual and peripheral blood NK cells. J Immunol 159:1071 23. Sivori S, Vitale M, Bottino C, Marcenaro E, Sanseverino L, Parolini S, Moretta L, Moretta A Ž1996. 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᎐2492 24. Carretero M, Cantoni C, Bellon T, Bottino C, Biassoni R, Rodriguez A, Perez-Villar JJ, Moretta L, Moretta A, LopezBotet M Ž1997. The CD94 and NKG2A C-type lectins covalently assemble to form a NK cell inhibitory receptor for HLA class I molecules. Eur J Immunol 27:563᎐567 25. Cantoni C, Biassoni R, Pende D, Sivori S, Accame L, Pareti L, Semenzato G, Moretta L, Moretta A, Bottino C Ž1998. The activating form of CD94 receptor complex. CD94 covalently associates with the Kp39 protein that represents the product of the NKG2-C gene. Eur J Immunol 28:327᎐338 26. Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G, Vitale C, Bertone S, Moretta A, Moretta L, Mingari MC Ž1998. Inhibitory receptors sensing HLA-G1 molecules in pregnancy: decidua-associated natural killer cells express LIR-1 and CD94rNKG2A and acquire p49, a novel HLA-G1-

specific receptor. Proc Natl Acad Sci USA Žin press. 27. Braud VM, Yvonne Jones E, McMichael A Ž1997. The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol 27:1164᎐1169 28. Braud VM, Allan DSJ, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, Lanier LL, McMichael AJ Ž1998. HLA-E binds to natural killer cell receptors CD94rNKG2A, B and C. Nature 391:795᎐799 29. Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez` Botet M, Gherarty D Ž1998. HLA-E is a major ligand for the natural killer inhibitory receptor CD94rNKG2A. Proc Natl Acad Sci USA 95:5199᎐5204 30. Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG Ž1998. Recognition of human histocompatibility leukocyte antigen ŽHLA.-E complexed with HLA class I signal sequence-derived peptides by CD94rNKG2 confers protection from Natural Killer cell- mediated lysis. J Exp Med 187:813᎐818 31. Cosman D, Fanger N, Borges L, Kubin M, Chin W, Peterson L, Hsu ML Ž1997. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273᎐282 32. Colonna M, Navarro F, Bellon ´ T, Llano M, Pilar G, Samaridis J, Angman L, Cella M, Lopez-Botet M Ž1997. A common ´ inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J Exp Med 186:1809᎐1818 33. Olcese L, Lang P, Vely ´ F, Cambiaggi A, Marguet D, Blery ´ M, Hippen KL, Biassoni R, Moretta A, Moretta L, Cambier JC, Vivier E Ž1996. Human and mouse Natural Killer cell inhibitory receptors recruit the PTP1C and PTP1D protein tyrosine phosphatase. J Immunol 156:4531᎐4534 34. Vitale W Castriconi R, Parolini S, Pende D, Hsu ML, Moretta L, Cosman D, Moretta A Ž1999. The LIR-1 receptor for the cytomegalovirus UL18 protein displays a broad specificity for different Hla class i alleles: analysis of LIR-1q natural killer cell clones. Int Immunol Žin press. 35. Cantoni C, Verdiani S, Falco M, Pessino A, Cilli M, Conte R, Pende D, Ponte M, Mikaelsson MS, Moretta L, Moretta A, Biassoni R Ž1998. p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily. Eur J Immunol 28:1980᎐1990 36. Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A Ž1997. p46, a novel Natural Killer cell-specific surface molecule which mediates cell activation. J Exp Med 186:1129᎐1136 37. Pessino A, Sivori S, Bottino C, Malaspina A, Morelli L, Moretta L, Biassoni R, Moretta A Ž1998. Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. J Exp Med 188:953᎐960 38. Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro R, Augugliaro R, Moretta L, Moretta A Ž1998. NKp44, a novel triggering surface molecule specifically expressed by activated Natural Killer cells is involved in nonMHC restricted tumor cell lysis. J Exp Med 187:2065᎐2072

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