Identification of an inhibitory MHC receptor on alloreactive rat natural killer cells.

Studies of allogeneic lymphocyte cytotoxicity have shown that the rat NK allorecognition repertoire is controlled by genetic elements in both the MHC (RT1) and the NK gene complex (NKC). DA rats, possessing NK cells that are unable to lyse allogeneic lymphoblasts, were immunized with alloreactive NK cells from MHC-matched PVG.1AV1 rats, and two mAb, STOK1 and STOK2, were generated. STOK1 and STOK2 stained identical subsets of NKR-P1+ T and NK cells from certain strains of rats. Relative numbers varied markedly in a panel of MHC congenic strains, however, implicating a role for self MHC genes in their development. Both STOK1 and STOK2 immunoprecipitated a 110-kDa disulfide-linked homodimeric molecule, with extensive N-linked glycosylations, encoded by a gene that mapped to the NKC. NK cells expressing this glycoprotein displayed an increased ability to lyse allogeneic lymphoblasts, while syngeneic targets were spared. However, blockade of the STOK2 Ag with F(ab')2 of STOK2 permitted the NK lysis of syngeneic targets, but did not affect NK allorecognition. These results indicate that mAb STOK1 and STOK2 identify an NKC-encoded MHC receptor in the rat that acts as a negative regulator of cytotoxicity.

Positive and negative MHC class I recognition by rat NK cells.

The prompt rejection of transplanted allogeneic lymphocytes by rat NK cells in non-sensitized recipients (allogeneic lymphocyte cytotoxicity or ALC) is determined by MHC genes as well as by genes located in the NK complex. The same genetic control is found when NK alloreactivity is measured by an in vitro assay, and we have employed this assay to delineate the specificity of NK cells for the MHC. The MHC of the rat, RT1, contains class I genes situated on either side of the class II/class III region. The majority of these class I genes are located in the RT1.C region and expressed class I products usually behave as non-classical (class Ib) molecules. They do not serve as restriction elements for the vast majority of conventional alpha/beta T-cells, in contrast to those class I molecules encoded by one or more loci in the classical (class Ia) region, RT1.A. However, NK cells appear to recognize the products of either class I region. Immunogenetic studies suggest that NK cells are inhibited by RT1. A molecules, whereas RT1.C region molecules may have a dual role in regulating NK cytolytic activity, i.e. they either inhibit or activate natural killing. Based on these premises, a model is proposed in which identification of a target as self or non-self depends on different receptors for class I in single NK cells, interpreting coincident positive and negative signals from the various target class I molecules. The putative role of peptides presented by class I, the biological implications, and the evolution of the NK receptors and their ligands are discussed.

Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition.

The cytolytic activity of human and mouse natural killer (NK) cells is negatively regulated by self major histocompatibility complex (MHC) class I molecules on potential target cells. In the rat, protection by RT1 class I gene products has so far not been formally shown although the complex effects of foreign and self RT1 genes on polyclonal NK cell activity suggest that MHC recognition can have both stimulatory and inhibitory effects. Here we report that the expression of self-MHC class I molecules on target cells strongly inhibits lysis by a long term NK cell line derived from LEW (RT1l) rats and by LEW NK cells activated by short-term culture in the presence of interleukin-2. This was demonstrated with mouse-rat hybridoma target cells expressing different rat MHC alleles and with mouse tumor target cells transfected with classical (RT1.Al) and nonclassical (RT1.Cl) rat MHC class I genes. With hybridoma target cells, the strongest reduction in lysis as compared to the parental mouse myeloma line was observed when "self" (LEW) MHC was expressed, while hybridomas expressing other MHC alleles showed less and variable reduction. Transfection of RT1.Al protected both L-929 fibroblasts and P815 mastocytoma cells from lysis by the NK cell line, while RT1.Cl only protected P815 cells, indicating that additional target cell properties regulate rat NK cell activity.

Alloreactive natural killer cells in the rat: complex genetics of major histocompatibility complex control.

A major role for the nonclassical major histocompatibility complex (MHC) class I region, i.e. RT1.C, in controlling rat natural killer (NK) cell alloreactivity has recently been established, and several findings suggested the existence of NK-triggering alloantigens coded for by this region. Here, we have extended our studies on the MHC control of NK cell cytotoxicity against concanavalin A-activated T cell blasts by comparing semi-syngeneic and fully allogeneic combinations, and we show the following: (a) The self MHC exerted a strong influence on the NK allorecognition repertoire. (b) When anti-F1 hybrid cytolytic activities of parental strain NK cells were measured, both recessively and non-recessively inherited susceptibility patterns emerged. (c) In most combinations parental strain cells were lysed by F1 hybrid NK cells, thus resembling the hybrid resistance phenomenon described in mice. The cytotoxicity was lower in strain combinations where NK susceptibility was inherited non-recessively, i.e. when parent anti-F1 reactivity was detected, than in recessive combinations. (d) LEW.1LM1 (RT1lm1) target cells, with a deletion in the RT1.C region that includes expressed class I genes, were more sensitive to lysis by MHC matched NK cells (PVG.1L(LEW), RT1l) than were parental LEW (RT1l) cells. The effect of the deletion was the opposite when MHC allogeneic (RT1c, RT1u) as well as semi-syngeneic (RT1l/c) NK cells were employed, i.e. sensitivity was decreased. We conclude that certain MHC-encoded antigens, depending on the haplotype combination of effector and target cells, may either trigger or inhibit rat NK cell cytotoxicity. Furthermore, the potential role of peptides bound to MHC class I molecules recognized by NK cells is discussed.

Control of rat natural killer cell-mediated allorecognition by a major histocompatibility complex region encoding nonclassical class I antigens.

The ability of natural killer (NK) cells to eliminate normal allogeneic hemic cells is well established in several species including mice, rats, and humans. The controlling elements for NK susceptibility in these species map to the major histocompatibility complex (MHC), but in contrast to findings in mice and humans, the mode of inheritance is not always recessive in rats. This finding is not easily explained by the missing self and hemopoietic histocompatibility (Hh) models for NK recognition, and has led to the idea that certain alloantigens may trigger NK cell reactivity. In our in vitro system for assessing rat NK alloreactivity, we have employed target and inhibitor cells from a large panel of MHC congenic, intra-MHC recombinant and MHC mutant rat strains, as well as appropriate F1 hybrids between them, and we show the following: (a) The nonclassical class I (RT1.C) region was most important in determining the susceptibility of target cells to alloreactive NK cells in vitro. Lymphocyte susceptibility to lysis in vivo also mapped to the C region, which supports the concept that the in vivo and in vitro alloreactivity assays reflect the same recognition process. (b) Four different RT1-controlled NK allospecificities (represented by the u, l, a, and n haplotypes) could be discerned when we used polyclonal NK cells from the PVG (RT1c) strain as effector cells. Three of the target specificities recognized were controlled mainly by the RT1.C region. (c) The expression of RT1.C region-controlled parental strain NK allodeterminants could be demonstrated in F1 hybrids heterozygous for the C region alone and were therefore inherited nonrecessively. (d) Loss of an RT1.C region-controlled NK allospecificity could be shown with the MHC mutant LEW.1LM1 rat strain characterized by a genomic deletion of about 100 kb of the C region. Taken together, these observations have demonstrated a major importance of the nonclassical class I region, i.e., RT1.C, in controlling rat NK allorecognition, and have thereby assigned a hitherto undescribed immunological property to this region. Furthermore, some of the present data are consistent with the existence of polymorphic NK-triggering alloantigens that are coded for by the RT1.C region.