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Biomedical subjects

M Llano

Publications and source records attributed to M Llano.

At least 19 recordsLinked to original sources
PubMed2001

Human cytomegalovirus and natural killer-mediated surveillance of HLA class I expression: a paradigm of host-pathogen adaptation.

Among various strategies to evade the host immune response, some viruses like human cytomegalovirus (HCMV) interfere with surface MHC class I expression and antigen presentation to T lymphocytes. The ability of natural killer (NK) cells to detect MHC class I molecules through inhibitory receptors can be envisaged as an adaptation of the immune system for responding to such pathological alterations. To fulfil that role, rodents use members of the Ly49 C-type lectin superfamily, whereas primates employ killer immunoglobulin-like receptors and the immunoglobulin-like transcript 2/leucocyte immunoglobulin-like receptor-1 receptor. CD94/NKG2 lectin-like heterodimers represent the most conserved receptor system for MHC class I molecules; by interacting with human HLA-E or murine Qa-1b, CD94/NKG2A inhibitory receptors broadly probe the biosynthesis pathway of other class I molecules. Reciprocally, HCMV has developed mechanisms to evade the NK response while modulating HLA class Ia expression. The ability of HCMV to maintain surface levels of HLA-E and to express an HLA class I surrogate (UL18) are herein discussed in the context of the interplay with human NKR systems.

Adaptation, Physiological↗
PubMed2001

Molecular studies and NK cell function of a new case of TAP2 homozygous human deficiency.

In this paper we describe the clinical and molecular features of a new case (GOR) of homozygous human TAP2 deficiency, analysing the phenotype and function of NK cells. The patient presented from infancy with recurrent sinopulmonary infections; a selective IgG2 deficiency, negative antibody response to polysaccharide vaccination and low level of cell surface expression of HLA class I antigens were found. The sequence of TAP2 gene identified a single mutation, a C to T substitution changing the CGA arg codon at amino acid 220 into TGA stop codon in exon 3. By using MoAbs for KIRs, CD94, CD94/NKG2A and ILT2 we observed, in agreement with others, that the latter two receptors were overexpressed on TAP2-deficient NK cells. The inhibitory CD94/NKG2A and triggering CD94/NKG2C NK receptors, specific for HLA-E, appeared to be functional in a limited number of NK clones that could be expanded in vitro. Expression of HLA-E was virtually undetectable in GOR B-LCL and very faint in PBMC, further supporting that interactions of class I leader sequence nonamers with HLA-E in the ER depend on a functional TAP complex.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing.

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK-target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK-target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

Animals↗
PubMed2000

Mitogen-activated protein kinase activity is involved in effector functions triggered by the CD94/NKG2-C NK receptor specific for HLA-E.

The CD94/NKG2C heterodimer constitutes an activating receptor involved in NK cell-mediated recognition of the class lb molecule HLA-E. It transduces the triggering signal through an ITAM-bearing molecule, DAP12/KARAP, coupled non-covalently to the receptor. Here we show that specific engagement of the receptor complex expressed on the surface of an NK clone induced the phosphorylation of mitogen-activated protein kinase (MAPK). By the use of the MEK inhibitor PD098059 we demonstrate that the MAPK pathway participates in the CD94-dependent TNF-alpha production and cytotoxicity. Moreover, we transferred the activating function by transfection of the heterologous RBL cell line with CD94/NKG2-C/DAP12. In this system, cross-linking of the receptor induced calcium mobilization, serotonin release and phosphorylation of MAPK.

Adaptor Proteins, Signal Transducing↗
PubMed2000

NK cell recognition of non-classical HLA class I molecules.

NK cells recognize several HLA class Ib molecules employing both immunoglobulin-like (Ig-like) and C-type lectin receptors. The CD94/NKG2 and NKG2D lectin-like molecules, respectively, interact with HLA-E and MICA; CD94/NKG2A functions as an inhibitory receptor, while CD94/NKG2C and NKG2D trigger NK cell activity. HLA-E predominantly presents nonamers from the leader sequences of other class I molecules; a peptide derived from HLA-G1 constitutes the highest affinity ligand for both CD94/NKG2 receptors. Members of the Ig-like transcript (ILT) or leucocyte Ig-like receptor (LIR) family (ILT2 or LIR-1 and ILT4 or LIR-2), expressed by other leucocyte lineages, interact with a broad spectrum of HLA class Ia molecules and HLA-G1. Among Ig-like KIRs, the KIR2DL4 (p49) receptor has been shown to specifically recognize HLA-G1; this molecule displays an unusual hybrid structure, sharing features with inhibitory and triggering KIRs.

Cytotoxicity, Immunologic↗
PubMed2000

Paired inhibitory and triggering NK cell receptors for HLA class I molecules.

Human natural killer (NK) cells specifically interact with major histocompatibility complex (MHC) class I molecules employing different receptor systems, shared with subsets of alphabeta and gammadelta T lymphocytes. Killer cell immunoglobulin-like receptors (KIRs) recognize groups of human leukocyte antigen (HLA) class Ia proteins displaying common structural features at the alpha-1 domain; among them, KIR2DL4 has been proposed to specifically interact with the class Ib molecule HLA-G1. Members of a related family of immunoglobulin (Ig)-like receptors (ILT2 or LIR-1 and ILT4 or LIR-2), expressed by other leukocyte lineages, interact with a broad spectrum of class Ia molecules and HLA-G1. On the other hand, CD94/NKG2-A(-C) and NKG2D lectin-like receptors, respectively, recognize the class Ib molecules HLA-E and MICA. A recurrent finding within the different receptor families is the existence of pairs of homologous molecules that often share the same ligands but display divergent functions. Inhibitory receptors tend to exhibit an affinity for HLA molecules higher than their activating counterparts. Recruitment of SH2 domain-bearing tyrosine phosphatases (SHP) by cytoplasmic phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIMs) is a crucial event for the inhibitory signalling pathway. By contrast, triggering receptors assemble with homodimers of immune tyrosine-based activation motif (ITAM)-bearing adaptor molecules (i.e., DAP12, CD3 xi) that engage tyrosine kinases (ZAP70 and syk).

Histocompatibility Antigens Class I↗
PubMed1999

Triggering of effector functions on a CD8+ T cell clone upon the aggregation of an activatory CD94/kp39 heterodimer.

Some T lymphocytes express the CD94 Ag, which is known to form heterodimers with members of the NKG2 family. We have studied the expression pattern and function of CD94 heterodimers in different alphabeta or gammadelta T cell clones. Most of the CD94+NKG2A- T cells have a low to intermediate expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer in one of these CD8 alphabeta CD94+NKG2A- T cell clones (K14B06) was able to: 1) increase the intracellular concentration of Ca2+, 2) induce the up-regulation of CD25 Ag expression and the secretion of IFN-gamma, and 3) trigger redirected cytotoxicity in a TCR-independent manner. This activatory property was not shared by any other costimulatory molecule expressed by the K14B06 T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The immunoprecipitation of CD94 heterodimer showed a 39-kDa band with a similar m.w. to the activatory heterodimer found on some NK clones. A novel form of the NKG2 family (NKG2H) was identified in K14B06. NKG2H protein represents an alternative spliced form of the NKG2E gene, displaying a charged residue in the transmembrane portion and a cytoplasmic tail that lacks immunoreceptor tyrosine-based inhibitory motifs. The expression of NKG2H in the cell membrane through its association to CD94 and DAP-12 molecules supports that it could form part of the activatory CD94/Kp39 heterodimer present on K14B06 cells.

Amino Acid Sequence↗
PubMed1999

The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells.

Previous studies on NK recognition of HLA-G1 employed as targets 721.221 transfectants (.221-G1) that unknowingly co-expressed the HLA-E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re-evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA-G1 by NK clones. We employed as targets .221-G1 cells and a surface HLA-E-negative transfectant, .221-G1(Eneg), generated by site-directed mutagenesis of the HLA-G1 leader sequence. The antagonistic effects of receptor- (ie. CD94/NKG2A, ILT2) and ligand-specific mAb (i.e. HLA-G, HLA-E) were assessed. In addition, binding of an ILT2-Ig fusion protein to the .221-AEH, expressing only HLA-E, and the .221-G1(Eneg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: the CD94/NKG2 interaction with HLA-E, and the engagement of the ILT2(LIR1) receptor by HLA-G1 molecules.

Antibodies, Monoclonal↗
PubMed1999

How do NK cells sense the expression of HLA-G class Ib molecules?

The nature of NK cell receptors involved in recognition of HLA-G1 has remained an unclear issue; we herein review this topic based on our experience. We found no evidence that well characterized p58 and p70 KIRs may interact with HLA-G1. By contrast, our data support that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: (1) the direct engagement of the ILT2 (LIR1) receptor by HLA-G1; and (2) the interaction of CD94/NKG2A and CD94/NKG2C receptors with the non-classical class I molecule HLA-E, co-expressed on the surface upon binding to a nonamer (VMAPRTLFL) from the HLA-G leader sequence.

Antigens, CD↗
PubMed1998

HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A.

We previously showed that the availability of a nonamer peptide derived from certain HLA class I signal sequences is a necessary requirement for the stabilization of endogenous HLA-E expression on the surface of 721.221 cells. This led us to examine the ability of HLA-E to protect HLA class I transfectants from natural killer (NK) cell-mediated lysis. It was possible to implicate the CD94/NKG2A complex as an inhibitory receptor recognizing this class Ib molecule by using as target a .221 transfectant selectively expressing surface HLA-E. HLA-E had no apparent inhibitory effect mediated through the identified Ig superfamily (Ig-SF) human killer cell inhibitory receptors or ILT2/LIR1. Further studies of CD94/NKG2+ NK cell-mediated recognition of .221 cells transfected with different HLA class I allotypes (i.e., -Cw4, -Cw3, -B7) confirmed that the inhibitory interaction was mediated by CD94/NKG2A recognizing the surface HLA-E molecule, because only antibodies directed against either HLA-E, CD94, or CD94/NKG2A specifically restored lysis. Surface stabilization of HLA-E in cold-treated .221 cells loaded with appropriate peptides was sufficient to confer protection, resulting from recognition of the HLA class Ib molecule by the CD94/NKG2A inhibitory receptor. Consistent with the prediction that the ligand for CD94/NKG2A is expressed ubiquitously, our examination of HLA-E antigen distribution indicated that it is detectable on the surface of a wide variety of cell types.

Antigens, CD↗
PubMed1998

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants.

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A+ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E+ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via Fc epsilonRI. Remarkably, interaction of CD94/NKG2-A+ RBL cells with the HLA-E+ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.

Animals↗
PubMed1998

HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer.

The HLA-E class Ib molecule constitutes a major ligand for the lectin-like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence-derived nonapeptides bind to endogenous HLA-E molecules in the HLA-defective cell line 721.221, inducing HLA-E surface expression, and promote CD94/NKG2A-mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA-E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA-E-bound peptides influences CD94/ NKG2-mediated recognition, beyond their ability to stabilize surface HLA-E. Further, CD94/ NKG2A+ NK clones appeared more sensitive to the interaction with most HLA-E-peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA-E loaded with the HLA-G-derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA-E/G-nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2-mediated recognition of HLA-E expressed on extravillous cytotrophoblasts plays an important role in maternal-fetal cellular interactions.

Antigens, CD↗
PubMed1997

A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells.

Natural killer (NK) cell-mediated lysis is negatively regulated by killer cell inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules. In this study, we characterize a novel inhibitory MHC class I receptor of the immunoglobulin-superfamily, expressed not only by subsets of NK and T cells, but also by B cells, monocytes, macrophages, and dendritic cells. This receptor, called Ig-like transcript (ILT)2, binds MHC class I molecules and delivers a negative signal that inhibits killing by NK and T cells, as well as Ca2+ mobilization in B cells and myelomonocytic cells triggered through the B cell antigen receptor and human histocompatibility leukocyte antigens (HLA)-DR, respectively. In addition, myelomonocytic cells express receptors homologous to ILT2, which are characterized by extensive polymorphism and might recognize distinct HLA class I molecules. These results suggest that diverse leukocyte lineages have adopted recognition of self-MHC class I molecules as a common strategy to control cellular activation during an immune response.

Amino Acid Sequence↗
PubMed1997

The CD94/NKG2-A inhibitory receptor complex is involved in natural killer cell-mediated recognition of cells expressing HLA-G1.

Human NK cells bear surface receptors that inhibit their cytolytic activity upon specific recognition of MHC class Ia Ags; little is known about the capacity of class Ib molecules to regulate NK cell function. We have studied the roles of different NK inhibitory receptors in recognition of the class Ib HLA-G. To this end, we analyzed the ability of an HLA-defective tumor cell line (721.221) transfected with the membrane form of HLA-G1, which contains the three external domains, to inhibit the cytolytic activity mediated by a panel of NK clones from several donors. A substantial proportion of peripheral blood NK clones appeared to be significantly inhibited by the HLA-G1-transfected cell line (referred to as .221-G1); nevertheless, no relation was observed between the expression and the function of serologically identifiable Ig-SF receptors (p58/p70) and specific recognition of .221-G1 cells. Moreover, p58 killer cell inhibitory receptor-IgG soluble fusion proteins specifically bound to 721.221 transfectants bearing their corresponding HLA-C ligands, but only a weak reactivity with .221-G1 cells was detectable. By contrast, most NK clones blocked by HLA-G1 expressed the CD94/NKG2-A inhibitory receptor, and moreover, CD94-specific mAbs reconstituted their cytolytic activity comparably to anti-HLA class I mAbs. These data support the idea that the CD94/NKG2 receptor complex is involved in the recognition of cells expressing HLA-G1.

Antibodies, Monoclonal↗
PubMed1997

The CD94/NKG2 C-type lectin receptor complex: involvement in NK cell-mediated recognition of HLA class I molecules.

A multigene family to human Ig-SF receptors and members of the murine Ly49 C-type lectin family are involved in natural killer (NK) cell-mediated recognition of MHC class I molecules. The human CD94 glycoprotein covalently assembles with different C-type lectins of the NKG2 family. By functional criteria, the CD94/NKG2-A (kp43) receptor complex appears also involved in NK cell-mediated recognition of different HLA class I allotypes. Similarly to the other NK inhibitory receptors, NKG2-A contains cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMS). By contrast, NK clones bearing different receptor complex (CD94/p39) are triggered upon ligation by CD94-specific monoclonal antibodies (MAbs); the p39 subunit is likely encoded by other member(s) of the NKG2 family. Expression of different CD94/NKG2 complexes is warranted to precisely assess their specific interaction with HLA class I molecules, and the molecular basis for their divergent functional properties.

Animals↗
PubMed1997

Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules.

A multigene family of immunoglobulin superfamily (Ig-SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H-2 products is mediated by members of the murine Ly49 C-type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C-type lectin receptor complex in NK cell-mediated recognition of Bw6+ HLA-B, HLA-A and HLA-C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glycoproteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig-SF receptor system. By contrast, NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94-specific mAbs. Remarkably, a set of Ig-SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94-associated molecules belong to the NKG2 family of C-type lectins; the NKG2-A gene encodes for the p43 subunit, which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.

Antigens, CD↗