MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus of day 14 fetal mice. These fetal NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although fetal NK cells are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.     

An allele-specific,stochastic gene expression process controls the expression of multiple Ly49family genes and generates a diverse,MHC-specific NK cell receptor repertoire

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the D ^d (or D^k )-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.     

The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1(u) haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49(+) and Ly49(-) NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.     

Location-specific regulation of transgenic Ly49A receptors by major histocompatibility complex class I molecules

Inhibitory receptors expressed on natural killer (NK) cells and T cells specific for major histocompatibility complex (MHC) class I are believed to prevent these cells from responding to normal self tissues. To understand the regulation and function of Ly49 receptor molecules in vivo, we used the CD2 promoter to target Ly49A expression to all thymocytes, T cells, and NK cells. In animals expressing its MHC class I ligand, H-2D^d or H-2D^k, there was a large decrease in the expression of Ly49A on thymocytes, peripheral T cells, and NK1.1⁺ cells. The extent of the down-regulation of Ly49A was dependent on the expression of the MHC ligand for Ly49A and on the site where the cells were located. The level of expression of endogenous Ly49A was similarly found to be dependent upon the organ where the cells resided. Data from bone marrow chimeras indicated that most cell types may regulate Ly49A expression, but the efficacy to regulate receptor expression may vary depending on the cell type.     

Loss or mismatch of MHC class I is sufficient to trigger NK cell-mediated rejection of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent pathways

A prediction from the "missing self" hypothesis is that down-regulation of MHC class I on resting hematopoietic cells should be sufficient to make them susceptible to NK cell killing. Using a method enabling kinetic and quantitative assessments of NK cell-mediated rejection responses in vivo, we here show that resting hematopoietic cells from beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice were rapidly rejected in unmanipulated C57BL/6 (B6) mice. In situations of allelic MHC class I mismatches rejection occurred but required longer time. beta(2)m(-/-) donor cells pre-activated with concanavalin A were more efficiently eliminated compared to resting cells, as were MHC(-) tumor cells. When recipient mice were pretreated with an IFN inducer to activate NK cells, rejection was also enhanced. The signaling adaptor KARAP/DAP12 was dispensable for rejection of beta(2)m(-/-) cells (lacking MHC) but critical for rejection of BALB/c cells (mismatched MHC) in unmanipulated B6 recipients. In contrast, B6 recipients with pre-activated NK cells rejected BALB/c cells in a KARAP/DAP12-independent fashion. Loss or mismatch of MHC class I in resting cells was thus sufficient to convey susceptibility to NK cell rejection. However, activation of the effector or the target enhanced rejection and shifted the balance between different signaling pathways involved.     

Expression of regulator of G protein signalling proteins in natural killer cells, and their modulation by Ly49A and Ly49D

The small GTPase accelerators regulator of G protein signalling (RGS) proteins are important regulators of proximal signalling from G protein coupled receptors. Although natural killer (NK) cells express a number of G-protein coupled receptors, expression of RGS proteins has not been investigated. We analysed the expression of RGS proteins in rat NK cells, and detected mRNA for RGS1, RGS2, RGS5, RGS8, RGS16, and RGS18. Interestingly, when we included a panel of different leucocyte subsets, we found that RGS8 was selectively expressed by NK cells. NK cells are under control of both activating and inhibitory receptors and, utilizing a xenogeneic system where the mouse activating Ly49D or inhibitory Ly49A receptors were transfected into the rat RNK-16 cell line, the potential regulation of RGS proteins by single NK cell receptors was studied. We found that ligation of Ly49D led to a rapid and transient increase in message for RGS2, while Ly49A ligation up-regulated RGS2, RGS16, and RGS18 mRNA. Both receptors also induced a prolonged increase in RGS2 endogenous protein levels. These findings suggest that RGS proteins may be influenced by or involved in NK cell receptor events, suggesting a crosstalk between G-protein coupled receptors and NK cell receptors.     

Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.     

Redundant functions of TCF-1 and LEF-1 during T and NK cell development,but unique role of TCF-1 for Ly49 NK cell receptor acquisition

Members of the TCF/LEF (T cell factor / lymphoid enhancer factor) family of DNA-binding factors play important roles during embryogenesis, the establishment and/or maintenance of self-renewing tissues such as the immune system and for malignant transformation. Specifically, it has been shown that TCF-1 is required for T cell development. A role for LEF-1 became apparent when mice harbored two hypomorphic TCF-1 alleles and consequently expressed low levels of TCF-1. Here we show that NK cell development is similarly regulated by redundant functions of TCF-1 and LEF-1, whereby TCF-1 contributes significantly more to NK cell development than LEF-1. Despite this role for NK cell development, LEF-1 is not required for the establishment of a repertoire of MHC class I-specific Ly49 receptors on NK cells. The proper formation of this repertoire depends to a large extent on TCF-1. These findings suggest common and distinct functions of TCF-1 and LEF-1 during lymphocyte development.     

Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region

CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.     

Co-evolution of MHC class I and variable NK cell receptors in placental mammals

Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests that KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of the functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines, a stepwise co-evolution of MHC class I and KIR is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C drove further elaboration of MHC-C-specific KIR, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor reportoire

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC-dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC-controlled repertoire differences are discussed.     

Cis association of leukocyte Ig‐like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18

Leukocyte Ig‐like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC‐I) and the human CMV MHC‐I homologue UL18. LIR‐1 can inhibit NK cells through the conventional interaction with MHC‐I expressed on a target cell (in trans) but the nature and the effects of LIR‐1 interactions with MHC‐I in cis are not well understood. Here we show that MHC‐I expressed in cis has an impact on the detection of LIR‐1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR‐1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC‐I alleles on 721.221 cells. We found that blocking the LIR‐1 contact site with anti‐MHC‐I antibodies decreased detection of LIR‐1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC‐I. Finally, disruption of LIR‐1 cis interactions with MHC‐I significantly enhanced UL18‐Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA‐G. These results have implications for LIR‐1 function in scenarios such as infection when MHC‐I levels on effector cells may be increased by IFNs.     

Stable expression of MHC class I heavy chain/HLA-DO complexes at the plasma membrane

Major histocompatibility complex (MHC) class I and II molecules present antigenic fragments to the immune system. MHC-like chaperones, like HLA-DM, HLA-DO and tapasin support peptide loading. HLA class I heavy chains require association with beta 2-microglobulin and peptide for endoplasmic reticulum (ER) exit. Likewise, HLA-DO is only able to leave the ER by association to DM. Here we show that HLA-DO and free MHC class I heavy chains associate into a stable complex early during biosynthesis and are expressed at the plasma membrane as a complex. These DO/heavy chain complexes are found on DO-transfected cells and on low amounts on human B cells.     

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k). The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

The MHC class I molecule H-2D^d (D^d) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered D^d molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the D^b molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (D^dS73WD156Y) impaired the protective effect of the D^d molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A⁺ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the α helix of the α2 domain, facing the solvent and without direct contact with the peptide (D^dA150S) had no effect. D^d recognition by Ly-49A⁺ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the D^d molecule that make the trimolecular complex less permissive for inhibition of Ly-49A⁺ NK cells.