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NKG2 receptors
Journal of Reproductive Immunology 43 (1999) 167 – 173 www.elsevier.com/locate/jreprimm
Review
NK cell mediated recognition of HLA class Ib molecules: role of CD94/NKG2 receptors Francisco Navarro, Manuel Llano, Pilar Garcı´a, Miguel Lo´pez-Botet * Ser6icio De InmunologI´a, Hospital Uni6ersitario De La Princesa, Diego de Leo´n 62, 28006 Madrid, Spain
Keywords: NK cell; CD94; HLA-E; HLA-G; Receptor – ligand interactions
It is currently accepted that the control of NK cell activity depends on a subtle balance between inhibitory and activating signals. NK cell functions are repressed upon specific recognition of MHC class I molecules (Ljunggren and Karre, 1990). Every mature NK cell is predicted to bear at least one dominant killer inhibitory receptor (KIR) for a self MHC class I product, thus preventing auto-reactivity against normal cells. On the other hand, the nature of receptor– ligand interactions triggering NK cell activity remains less defined; yet, some activating molecules are structurally related to the MHC class I-specific KIRs. Conventional experimental systems analyse the activity of NK clones against HLA class I-defective tumor cell lines of hematopoietic origin transfected with individual class I allotypes. Specific recognition leads to the inhibition of cytotoxicity, which can be reconstituted by the antagonistic effect of mAbs directed against either MHC molecules or KIRs. Multiple
Presented at the First International Conference on HLA-G, Paris, July 1998. * Corresponding author. Tel.: + 34-91-5202386; fax: + 34-91-3092496. E-mail address: mlbotet/[email protected] (M. Lo´pez-Botet)
0165-0378/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 3 0 - 3
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combinations of NK cell receptors (NKR) may be co-expressed by individual NK clones, thus often rendering difficult the dissection of their individual role in cellular assays. The frequency of cells expressing each receptor is variable among different individuals, and there are indications suggesting that the NKR repertoire is influenced by genetic factors (Uhrberg et al., 1997). A group of human KIRs are encoded by an Immunoglobulin (Ig)-related multigene family located in chromosome 19, and each receptor specifically interacts with a different set of HLA class I allotypes (Colonna and Samaridis, 1995; Wagtmann et al., 1995; Lanier, 1997; Moretta and Moretta, 1997). Formal proofs for the direct interaction of Ig-SF KIRs with HLA molecules have been obtained, and the crystal structure of a p58 KIR has been defined (Long and Wagtmann, 1997). A common structural feature of KIRs is the presence of cytoplasmic ‘immunoreceptor tyrosinebased inhibitory motifs’ (ITIMs, V/I/LxYxxL/V sequences) which, upon tyrosine phosphorylation, recruit protein tyrosine phosphatases (i.e. SHP-1) involved in the down-regulation of NK cell activity (Leibson, 1997; Vivier and Daeron, 1997). Remarkably, other members of the Ig-SF NK receptor (NKR) family can trigger NK cell-mediated lysis upon their ligation. These activating receptors are closely homologous to inhibitory molecules in their extracellular region, but contain shorter intracytoplasmic domains lacking ITIMs, and display a different transmembrane sequence (Moretta and Moretta, 1997). The non-inhibitory receptors establish electrostatic interactions with dimers DAP-12, an ITAM-bearing 12 kDa protein similar to the z chain of the TCR involved in signalling, also termed KARAP (Olcese et al., 1997; Lanier et al., 1998). All known NKR are detected on ab and gd T-lymphocyte subsets, regulating T-cell receptor (TCR)-mediated and NK-like cytotoxicity (Moretta et al., 1996) . Recently, a related family of Ig-like receptors, some of which interact with HLA class I molecules, has been identified in different leukocyte lineages, including B lymphocytes and myelomonocytic cells. These observations support that recognition of MHC class I molecules represents a general regulatory strategy for the Immune System (Colonna et al., 1997). Notably, one of this inhibitory receptors (ILT2), broadly reactive with different HLA class I molecules, has been shown to bind also the UL18 glycoprotein, an HLA class I homologue from human cytomegalovirus (Cosman et al., 1997). In rodents, recognition of H-2 products is mediated by members of the Ly49 C-type lectin family (Yokoyama, 1995). Several type II integral membrane glycoproteins (CD94, NKG2 and hNKR-P1A), that contain an extracellular C-type carbohydrate recognition domain (CRD) and display partial homology with the murine NK gene complex (NKC) families
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(NKR-P1 and Ly49), have been identified in human NK cells, and are encoded by genes clustered in chromosome 12. The CD94 glycoprotein covalently assembles with distinct C-type lectins of the NKG2 family; CD94 is expressed by NK cells and a subset of T lymphocytes (Lazetic et al., 1996; Carretero et al., 1997; Lo´pez-Botet et al., 1998). The CD94/NKG2A heterodimer constitutes an inhibitory receptor coupled to SHP phosphatases; by contrast, the association of CD94 with the NKG2C protein, highly homologous to NKG2A but lacking ITIM, forms a receptor with triggering function (Pe´rez-Villar et al., 1996; Houchins et al., 1997; Lo´pezBotet et al., 1998; Cantoni et al., 1998). Additional putative triggering receptors with a different specificity may be assembled (i.e. CD94/NKG2E). The activating role of the CD94/NKG2-C dimer also linked to DAP12/ KARAP, points out a remarkable resemblance between Ig-SF and C-type lectin (CD94/NKG2) NKR systems; in both instances, pairs of molecules closely homologous in the extracellular region can fulfill divergent functions. CD94 homologues have been identified in mouse (Vance et al., 1997) and rat (Dissen et al., 1997), whereas the existence of human Ly49 molecules remains uncertain. Based on indirect functional criteria, CD94/NKG2A was originally considered to be involved in specific NK cell-mediated recognition of a broad spectrum of HLA class Ia allotypes (Moretta et al., 1994; Phillips et al., 1996; Sivori et al., 1996) and the HLA-G1 class Ib molecule (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997); yet, according to recent observations this concept has changed. In this regard, it has been formally established that the HLA-E class Ib molecule is specifically recognized by the CD94/NKG2 receptor complex (Borrego et al., 1998; Braud et al., 1998; Carretero et al., 1998; Lee et al., 1998b). Endogenous HLA-E molecules are stabilized on the surface of the 721.221 HLA-defective target cells upon transfection with HLA allotypes which contain within their leader sequences nonapeptides capable of assembling with the HLA-E a chain by a TAP-dependent mechanism (Braud et al., 1997; Lee et al., 1998a); nonamers containing a Thr rather than a Met in P2 do not significantly stabilize the expression of HLA-E. Furthermore, the selective property of several transfected HLA class Ia molecules to protect the 721.221 cell line against CD94/NKG2-A +NK cells actually depends on their ability to concomitantly induce surface expression of endogenous HLA-E, thus suggesting that this class Ib molecule is a major if not the sole ligand for the C-type lectin receptor complex (Braud et al., 1998; Lee et al., 1998b). Our recent studies show that the structure of nonamers presented by HLA-E may influence CD94/NKG2A and CD94/NKG2C-mediated recognition, beyond their ability to bind and stabilize surface expression of the
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HLA class Ib molecule (Llano et al., 1998). In particular, the presence of a non-polar residue in P6, corresponding to the leader sequence of several HLA-Cw*07 subtypes stabilized HLA-E but was inefficiently recognized by CD94/NKG2 receptors. Interestingly, the HLA-G1-derived nonamer bound to HLA-E constituted in these experiments a ligand for both CD94/NKG2 receptors. The capacity of inhibitory and triggering CD94/NKG2 receptors to discriminate among different HLA-E/peptide complexes may be important for NK/T cell reactivity against allogeneic and virus-infected cells. The recent resolution of the crystal structure of HLA-E (O’Callaghan et al., 1998) will enable a more precise molecular approach to these issues. The possibility that CD94/NKG2 might directly recognize HLA-G1 molecules was proposed on the basis of indirect functional assays using transfected.221 cells, prior to the knowledge on the role of HLA-E coexpression (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997). However, the leader sequence of HLA-G contains a nonamer capable of binding to the HLA-E molecule; in fact, surface expression of the latter is stabilized upon transfection of HLA-G in.221 cells. Thus, we addressed whether CD94/NKG2 might interact not only with HLA-E but also with HLA-G. To this end we analyzed the effect of HLA-G, HLA-E and NKR-specific mAbs in functional assays with NK clones as effectors tested against the.221-G1 cell line that co-expressed HLA-E. Moreover, by site directed mutagenesis of the HLA-G1 leader sequence we generated and used as target a 221-G1 transfectant that did not co-express surface HLA-E. Our results with this approach (Navarro et al., 1999) support that NK recognition of.221 cells expressing HLA-G1 involves at least two distinct non-overlapping receptor-ligand systems: the CD94/NKG2-HLA-E interaction, and a direct engagement of HLA-G1 molecules by the broadly reactive ILT2/ LIR1 receptor; a formal proof for binding of the latter to HLA-G1 was already reported (Colonna et al., 1997). While the proposed participation of the known Ig-SF KIRs in recognition of HLA-G (Pazmany et al., 1996; Mandelboim et al., 1997; Munz et al., 1997) still remains controversial (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997), the existence of additional NKR interacting with HLA-G is not excluded. Regulation of surface HLA-E expression in trophoblast cells, where it can be detected by immunohistochemical studies with the 3D12 mAb (D. Geraghty, personal communication), presumably depends mainly on HLAG biosynthesis, as this appears to be the predominant class I molecule in that tissue; yet, a contribution of HLA-C-derived nonamers is also possible (Carosella et al., 1996; King et al., 1997). The efficient CD94/NKG2-mediated recognition of HLA-E molecules bound to the HLA-G leader sequence-derived nonamer supports a specific functional role for the C-type lectin receptor complex in the context of the materno–fetal interaction, that
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deserves attention. In this regard, it is of note that most decidual NK cells express the CD94/NKG2A inhibitory receptor and, moreover, also NKG2C transcripts have been detected by RT-PCR in these samples (A. King, personal communication).
Acknowledgements This work was supported by grants SAF96/0335 (Plan Nacional I+ D), PL950062 (European Commission) and 07/055/96 (C.A.M.).
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Review
NK cell mediated recognition of HLA class Ib molecules: role of CD94/NKG2 receptors Francisco Navarro, Manuel Llano, Pilar Garcı´a, Miguel Lo´pez-Botet * Ser6icio De InmunologI´a, Hospital Uni6ersitario De La Princesa, Diego de Leo´n 62, 28006 Madrid, Spain
Keywords: NK cell; CD94; HLA-E; HLA-G; Receptor – ligand interactions
It is currently accepted that the control of NK cell activity depends on a subtle balance between inhibitory and activating signals. NK cell functions are repressed upon specific recognition of MHC class I molecules (Ljunggren and Karre, 1990). Every mature NK cell is predicted to bear at least one dominant killer inhibitory receptor (KIR) for a self MHC class I product, thus preventing auto-reactivity against normal cells. On the other hand, the nature of receptor– ligand interactions triggering NK cell activity remains less defined; yet, some activating molecules are structurally related to the MHC class I-specific KIRs. Conventional experimental systems analyse the activity of NK clones against HLA class I-defective tumor cell lines of hematopoietic origin transfected with individual class I allotypes. Specific recognition leads to the inhibition of cytotoxicity, which can be reconstituted by the antagonistic effect of mAbs directed against either MHC molecules or KIRs. Multiple
Presented at the First International Conference on HLA-G, Paris, July 1998. * Corresponding author. Tel.: + 34-91-5202386; fax: + 34-91-3092496. E-mail address: mlbotet/[email protected] (M. Lo´pez-Botet)
0165-0378/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 3 0 - 3
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combinations of NK cell receptors (NKR) may be co-expressed by individual NK clones, thus often rendering difficult the dissection of their individual role in cellular assays. The frequency of cells expressing each receptor is variable among different individuals, and there are indications suggesting that the NKR repertoire is influenced by genetic factors (Uhrberg et al., 1997). A group of human KIRs are encoded by an Immunoglobulin (Ig)-related multigene family located in chromosome 19, and each receptor specifically interacts with a different set of HLA class I allotypes (Colonna and Samaridis, 1995; Wagtmann et al., 1995; Lanier, 1997; Moretta and Moretta, 1997). Formal proofs for the direct interaction of Ig-SF KIRs with HLA molecules have been obtained, and the crystal structure of a p58 KIR has been defined (Long and Wagtmann, 1997). A common structural feature of KIRs is the presence of cytoplasmic ‘immunoreceptor tyrosinebased inhibitory motifs’ (ITIMs, V/I/LxYxxL/V sequences) which, upon tyrosine phosphorylation, recruit protein tyrosine phosphatases (i.e. SHP-1) involved in the down-regulation of NK cell activity (Leibson, 1997; Vivier and Daeron, 1997). Remarkably, other members of the Ig-SF NK receptor (NKR) family can trigger NK cell-mediated lysis upon their ligation. These activating receptors are closely homologous to inhibitory molecules in their extracellular region, but contain shorter intracytoplasmic domains lacking ITIMs, and display a different transmembrane sequence (Moretta and Moretta, 1997). The non-inhibitory receptors establish electrostatic interactions with dimers DAP-12, an ITAM-bearing 12 kDa protein similar to the z chain of the TCR involved in signalling, also termed KARAP (Olcese et al., 1997; Lanier et al., 1998). All known NKR are detected on ab and gd T-lymphocyte subsets, regulating T-cell receptor (TCR)-mediated and NK-like cytotoxicity (Moretta et al., 1996) . Recently, a related family of Ig-like receptors, some of which interact with HLA class I molecules, has been identified in different leukocyte lineages, including B lymphocytes and myelomonocytic cells. These observations support that recognition of MHC class I molecules represents a general regulatory strategy for the Immune System (Colonna et al., 1997). Notably, one of this inhibitory receptors (ILT2), broadly reactive with different HLA class I molecules, has been shown to bind also the UL18 glycoprotein, an HLA class I homologue from human cytomegalovirus (Cosman et al., 1997). In rodents, recognition of H-2 products is mediated by members of the Ly49 C-type lectin family (Yokoyama, 1995). Several type II integral membrane glycoproteins (CD94, NKG2 and hNKR-P1A), that contain an extracellular C-type carbohydrate recognition domain (CRD) and display partial homology with the murine NK gene complex (NKC) families
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(NKR-P1 and Ly49), have been identified in human NK cells, and are encoded by genes clustered in chromosome 12. The CD94 glycoprotein covalently assembles with distinct C-type lectins of the NKG2 family; CD94 is expressed by NK cells and a subset of T lymphocytes (Lazetic et al., 1996; Carretero et al., 1997; Lo´pez-Botet et al., 1998). The CD94/NKG2A heterodimer constitutes an inhibitory receptor coupled to SHP phosphatases; by contrast, the association of CD94 with the NKG2C protein, highly homologous to NKG2A but lacking ITIM, forms a receptor with triggering function (Pe´rez-Villar et al., 1996; Houchins et al., 1997; Lo´pezBotet et al., 1998; Cantoni et al., 1998). Additional putative triggering receptors with a different specificity may be assembled (i.e. CD94/NKG2E). The activating role of the CD94/NKG2-C dimer also linked to DAP12/ KARAP, points out a remarkable resemblance between Ig-SF and C-type lectin (CD94/NKG2) NKR systems; in both instances, pairs of molecules closely homologous in the extracellular region can fulfill divergent functions. CD94 homologues have been identified in mouse (Vance et al., 1997) and rat (Dissen et al., 1997), whereas the existence of human Ly49 molecules remains uncertain. Based on indirect functional criteria, CD94/NKG2A was originally considered to be involved in specific NK cell-mediated recognition of a broad spectrum of HLA class Ia allotypes (Moretta et al., 1994; Phillips et al., 1996; Sivori et al., 1996) and the HLA-G1 class Ib molecule (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997); yet, according to recent observations this concept has changed. In this regard, it has been formally established that the HLA-E class Ib molecule is specifically recognized by the CD94/NKG2 receptor complex (Borrego et al., 1998; Braud et al., 1998; Carretero et al., 1998; Lee et al., 1998b). Endogenous HLA-E molecules are stabilized on the surface of the 721.221 HLA-defective target cells upon transfection with HLA allotypes which contain within their leader sequences nonapeptides capable of assembling with the HLA-E a chain by a TAP-dependent mechanism (Braud et al., 1997; Lee et al., 1998a); nonamers containing a Thr rather than a Met in P2 do not significantly stabilize the expression of HLA-E. Furthermore, the selective property of several transfected HLA class Ia molecules to protect the 721.221 cell line against CD94/NKG2-A +NK cells actually depends on their ability to concomitantly induce surface expression of endogenous HLA-E, thus suggesting that this class Ib molecule is a major if not the sole ligand for the C-type lectin receptor complex (Braud et al., 1998; Lee et al., 1998b). Our recent studies show that the structure of nonamers presented by HLA-E may influence CD94/NKG2A and CD94/NKG2C-mediated recognition, beyond their ability to bind and stabilize surface expression of the
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HLA class Ib molecule (Llano et al., 1998). In particular, the presence of a non-polar residue in P6, corresponding to the leader sequence of several HLA-Cw*07 subtypes stabilized HLA-E but was inefficiently recognized by CD94/NKG2 receptors. Interestingly, the HLA-G1-derived nonamer bound to HLA-E constituted in these experiments a ligand for both CD94/NKG2 receptors. The capacity of inhibitory and triggering CD94/NKG2 receptors to discriminate among different HLA-E/peptide complexes may be important for NK/T cell reactivity against allogeneic and virus-infected cells. The recent resolution of the crystal structure of HLA-E (O’Callaghan et al., 1998) will enable a more precise molecular approach to these issues. The possibility that CD94/NKG2 might directly recognize HLA-G1 molecules was proposed on the basis of indirect functional assays using transfected.221 cells, prior to the knowledge on the role of HLA-E coexpression (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997). However, the leader sequence of HLA-G contains a nonamer capable of binding to the HLA-E molecule; in fact, surface expression of the latter is stabilized upon transfection of HLA-G in.221 cells. Thus, we addressed whether CD94/NKG2 might interact not only with HLA-E but also with HLA-G. To this end we analyzed the effect of HLA-G, HLA-E and NKR-specific mAbs in functional assays with NK clones as effectors tested against the.221-G1 cell line that co-expressed HLA-E. Moreover, by site directed mutagenesis of the HLA-G1 leader sequence we generated and used as target a 221-G1 transfectant that did not co-express surface HLA-E. Our results with this approach (Navarro et al., 1999) support that NK recognition of.221 cells expressing HLA-G1 involves at least two distinct non-overlapping receptor-ligand systems: the CD94/NKG2-HLA-E interaction, and a direct engagement of HLA-G1 molecules by the broadly reactive ILT2/ LIR1 receptor; a formal proof for binding of the latter to HLA-G1 was already reported (Colonna et al., 1997). While the proposed participation of the known Ig-SF KIRs in recognition of HLA-G (Pazmany et al., 1996; Mandelboim et al., 1997; Munz et al., 1997) still remains controversial (Pende et al., 1997; Pe´rez-Villar et al., 1997; So¨derstro¨m et al., 1997), the existence of additional NKR interacting with HLA-G is not excluded. Regulation of surface HLA-E expression in trophoblast cells, where it can be detected by immunohistochemical studies with the 3D12 mAb (D. Geraghty, personal communication), presumably depends mainly on HLAG biosynthesis, as this appears to be the predominant class I molecule in that tissue; yet, a contribution of HLA-C-derived nonamers is also possible (Carosella et al., 1996; King et al., 1997). The efficient CD94/NKG2-mediated recognition of HLA-E molecules bound to the HLA-G leader sequence-derived nonamer supports a specific functional role for the C-type lectin receptor complex in the context of the materno–fetal interaction, that
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deserves attention. In this regard, it is of note that most decidual NK cells express the CD94/NKG2A inhibitory receptor and, moreover, also NKG2C transcripts have been detected by RT-PCR in these samples (A. King, personal communication).
Acknowledgements This work was supported by grants SAF96/0335 (Plan Nacional I+ D), PL950062 (European Commission) and 07/055/96 (C.A.M.).
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