Jonas Sundbäck

Host MHC class I gene control of NK-cell specificity in the mouse

Summary: The missing self model predicts chat NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host, Transgenic and gene knock‐out mice have provided conclusive evidence chat MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the POSSIBLE Mechanisms behind it. starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK‐cell functional specificity as well as Lγ49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain “useful” NK cells, while avoiding “harmful” and “useless” ones? The available data can be fitted with in each of two mutually non‐exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as energy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers.

Murine natural killer (NK) cells are inhibited from killing their targets by the interaction between inhibitory, C‐type lectin like Ly49 receptors and major histocompatibility complex (MHC) class I molecules. The receptors have overlapping specificity, and it has been difficult to analyze specific aspects of the interaction between different Ly49 receptors and their respective ligands. We have addressed this problem using tetramers of bacterially expressed, non‐glycosylated, MHC class I molecules refolded with different peptides. Our results indicate that this technology is useful for analysis of Ly49 receptor specificity as well as for monitoring of NK cell subsets, with the following major conclusions emerging from this study: (1) tetramers of H‐2Dd bound the Ly49A receptor; the MHC associated glycan, previously suggested to be involved in recognition by this receptor, is thus not required for Ly49A receptor binding; (2) in support and extension of a recent report indicating peptide selectivity in the recognition of H‐2Kb by Ly49C+ cells, H‐2Kb tetramer binding to Ly49C receptors was strongly influenced by the peptide presented by the MHC class I molecule; (3) tetramer binding allowed visualization of interactions that have not previously been detected in functional studies, such as the recognition of H‐2Db by Ly49A and Ly49C.

T Cell Tolerance Based on Avidity Thresholds Rather Than Complete Deletion Allows Maintenance of Maximal Repertoire Diversity

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1α (EF1α). EF1α occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1α peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1α peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1α isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.

PEPTIDE DEPENDENCY AND SELECTIVITY OF THE NK CELL INHIBITORY RECEPTOR LY-49C

MHC class I molecules can prevent NK cell‐mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin‐superfamily receptors, we have studied a system based on the murine NK receptor Ly‐49C in the lectin‐superfamily. Loading of TAP‐deficient RMA‐S cells with the H‐2Kb‐restricted, ovalbumin‐derived peptide OVA257 – 264 (pOVA) induced their ability to bind Ly‐49C‐transfected reporter cells, and also protected them from killing by Ly‐49C+ NK cells. Other peptides that bound and stabilized H‐2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C‐terminal parts of the α1 helix (73 – 77) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73 – 77. In conclusion, protection mediated by the murine C‐type lectin receptor Ly‐49C is peptide dependent and selective.

Murine class I major histocompatibility complex H–2Dd: expression, refolding and crystallization

A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system. It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein. Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions. The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution.

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‐2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly‐49A. We have constructed altered Dd molecules by site‐directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly‐49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd 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 (DdA150S) had no effect. Dd 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 Dd molecule that make the trimolecular complex less permissive for inhibition of Ly‐49A+ NK cells.

NK Cell Inhibitory Receptor Ly-49C Residues Involved in MHC Class I Binding

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2Dd, as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2Kb, H-2Db, and H-2Dd. The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2Dd in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.

Interference with O-glycosylation in RMA lymphoma cells leads to a reduced in vivo growth of the tumor

Carbohydrate processing in cancer cells can influence the growth, metastatic potential, vascularization and immune recognition of such cells. Interference with N‐glycosylation has been shown both to reduce the membrane expression of MHC class I and to increase the in vitro sensitivity of tumor cells to NK cell killing. We investigated the effect of O‐glycosylation inhibition on the in vivo growth, phenotype and NK sensitivity of RMA lymphoma cells using benzyl N‐acetyl‐α‐D‐galactosamide (BAG). BAG‐treated cells were found to have a strongly reduced local growth potential in vivo. However, inhibition of O‐glycosylation caused this effect without any significant downregulation of MHC‐I and increase in sensitivity to NK killing as seen after inhibition of N‐glycosylation using Castanospermine. BAG treatment of RMA cells resulted in the removal of larger O‐linked glycans and a high expression of the T‐antigen (GalGalNAc), a target for natural antibodies (NAs) induced by the gastrointestinal bacterial flora. Whether the loss of larger O‐linked glycans, and associated functions, or of biological effects of NA contributed to the antitumor effect remains to be established. The results support the idea that inhibitors of O‐ as well as N‐linked glycosylation may be useful for the treatment of cancer, given that they can be specifically targeted to the tumor tissue.