Ly49 and CD94/NKG2: developmentally regulated expression and evolution.

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.

The non-classical MHC class I molecule Qa-1(b) inhibits classical MHC class I-restricted cytotoxicity of cytotoxic T lymphocytes.

The CD94/NKG2A heterodimer is an inhibitory receptor expressed on a subset of mouse NK cells. CD94/NKG2A recognizes the non-classical MHC class I (class Ib) molecule Qa-1(b) and inhibits NK cytotoxicity. Qa-1(b) presents a peptide derived from the leader sequence of classical MHC class I molecules. Here, we examined the role of CD94/NKG2A in T cell-mediated cytotoxicity. Soluble tetrameric Qa-1(b) bound to almost all CD8(+), but not CD4(+), T cells. This binding seems to be mediated by CD8, because COS cells transfected with CD8 also bound Qa-1(b) tetramer. Therefore, the expression of CD94/NKG2 in T cells was further examined by single-cell RT-PCR. Most murine CD8(+) T cells constitutively expressed CD94 and NKG2A transcripts, whereas they were not detected in CD4(+) T cells. Co-expression of Qa-1(b) and D(k) on target cells significantly inhibited cytotoxicity of D(k)-specific cytotoxic T lymphocytes generated by mixed lymphocyte reaction, indicating that Qa-1(b) on antigen-presenting cells interacts with CD94/NKG2A on CD8 T cells and regulates classical MHC class I-restricted cytotoxic T cells. These results suggest a significant role of CD94/NKG2A as an inhibitory receptor on CD8(+) T cells.

IFN-gamma production and cytotoxicity of IL-2-activated murine NK cells are differentially regulated by MHC class I molecules.

Activation of NK cells by target cells leads to cytotoxicity as well as production of various cytokines including IFN-gamma. MHC class I molecules on target cells regulate NK cytotoxicity. However, little is known about the regulation of IFN-gamma production by NK cells. We examined the production of IFN-gamma in individual murine NK cells stimulated with tumor cell lines by flow cytometric analysis of intracellular IFN-gamma. Among several tumor lines tested, the rat basophilic leukemia line RBL-1 induced particularly high level of IFN-gamma production in IL-2-activated NK cells, whereas other lines, including the prototypic NK target YAC-1, induced very low or no IFN-gamma production. Transfection of murine classical MHC class I molecules into RBL-1 cells substantially inhibited IFN-gamma production. This inhibition of IFN-gamma production by MHC class I was independent of Ly-49 or CD94/NKG2A expression on NK cells. These results indicate that some target cells directly stimulate IL-2-activated NK cells and induce IFN-gamma production, but the requirements for the induction of IFN-gamma production seem different from those for NK cytotoxicity. Furthermore, similar to NK cytotoxicity, induction of IFN-gamma production is inhibited by MHC class I on stimulating cells. However, the MHC class I-specific receptors inhibiting IFN-gamma production are different from those for NK cytotoxicity.

Recognition of class I MHC by NK receptor Ly-49C: identification of critical residues.

The Ly-49 family of inhibitory receptors plays a major role in regulating mouse NK cell cytotoxicity. Two of its members, Ly-49C and I, are recognized by the mAb 5E6, which also defines a subset of NK cells involved in the hybrid resistance phenomenon. Previous studies have shown that Ly-49C binds to a broad spectrum of class I MHC molecules, while Ly-49I apparently does not bind to any class I MHC molecules tested. In the present investigation we have defined the amino acid residues of Ly-49C that are critical for determining its ligand specificities. First, using quantitative COS cell adhesion assays, we demonstrated that Ly-49CB6 bound to Dd, Db, Kb, or Kk as well as to murine leukemic cell lines GM979 (H-2s) and IC-21 (H-2b). In contrast, COS cells expressing Ly-49IB6 did not significantly bind to any of the class I MHC tested. To determine which amino acid residues of Ly-49C are critical for their specific binding to class I MHC, a series of chimeric and mutant Ly-49C and I were generated and tested. Exchanging the critical residues between Ly-49C and I significantly affected their binding specificities. Finally, we identified the epitopes on Ly-49C recognized by mAbs 5E6 and 4LO3311 that functionally inhibit Ly-49C recognition of its ligands. These results further define the class I specificities of Ly-49C and provide insight into the structural basis for how class I MHC is recognized by the Ly-49 family of NK receptors.

Dextran sulfate inhibits IFN-gamma-induced Jak-Stat pathway in human vascular endothelial cells.

Human vascular endothelial cells can be induced by IFN-gamma to express class II MHC proteins. Previously, dextran sulfate was shown to selectively inhibit expression of class II MHC by preventing transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. In this study we characterized the effects of dextran sulfate on the intracellular events occurring prior to CIITA activation. Immunoprecipitation and Western blot analyses indicated that IFN-gamma-induced phosphorylation of Stat1 and Jak2 was blocked by dextran sulfate. In addition, electron micrographs showing the large accumulation of dextran sulfate particles in the cytoplasms of endothelial cells demonstrated that Stat and Jak proteins may directly interact with dextran sulfate. Binding of radiolabeled IFN-gamma to cells indicated that dextran sulfate may also modulate IFN-gamma interactions with the cell surface. Thus, dextran sulfate is capable of interfering with the IFN-gamma-induced expression of class II MHC genes at multiple sites.

Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor.

The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.

IFN-gamma-induced MHC class II gene expression is suppressed in endothelial cells by dextran sulfate.

IFN-gamma-activated endothelial cells actively participate in initiating immune responses by interacting with immunocompetent cells via class II MHC proteins. In this study, dextran sulfate, a synthetic heparin analogue, was shown to selectively inhibit IFN-gamma-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells, but not other cytokine-induced molecules such as ELAM-1 or ICAM-1. Inhibition occurred regardless of whether dextran sulfate was added 24 h before, at the same time as, or 24 h after IFN-gamma stimulation of cells. In addition, both high (500 kDa) and low (5 kDa) molecular mass dextran sulfate molecules were able to block class II expression, whereas treating cells with naturally occurring polysulfated glycosaminoglycans such as heparin, heparan, and chondroitin sulfate did not produce any suppressive effects. Radiolabeling of cells with [35S]methionine followed by radioimmunoprecipitation using anti-HLA-DR alpha mAb demonstrated that biosynthesis of class II proteins was specifically blocked. RT-PCR and Southern blotting were utilized to examine transcription of the HLA-DR alpha gene and demonstrated an absence of HLA-DR alpha mRNA from dextran sulfate-treated and IFN-gamma-induced cells. Dextran sulfate also prevented transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. Thus, dextran sulfate apparently inhibited this step or an earlier one in the intracellular signaling pathway for IFN-gamma in human endothelial cells, subsequent to IFN-gamma binding to its cell surface receptor.