The effect of in vivo depletion of NKR-P1+ or CD8+ lymphocytes on the acute rejection of allogeneic lymphocytes (ALC) in the rat.

We have depleted lymphocyte subsets in PVG and AO rats with MoAbs 3.2.3 (against NKR-P1 on NK and NK/T cells) and OX-8 (against CD8 on CTL and NK cells), and examined the effect on the killing of YAC-1 target cells in vitro and the effect on the acute rejection of small allogeneic lymphocytes in vivo (allogeneic lymphocyte cytotoxicity, ALC). While 3.2.3 treatment led to only a partial depletion of 3.2.3-positive cells in PVG rats, this treatment drastically reduced the number of NKR-P1+ cells in AO rats, abolished splenic NK activity against the NK-sensitive tumour target YAC-1, and markedly diminished the ALC response. Rats treated with OX-8 for 1 day showed a similar loss of NK cell function in vivo and in vitro. However, in rats treated with OX-8 for 3 days a 3.2.3+ and OX-8- population consisting of NK cells appeared, restoring ALC. The results demonstrate that NK cell responses can be greatly diminished after in vivo treatment with these MoAbs. Furthermore, they demonstrate that ALC is not necessarily linked to expression of the CD8 molecule.

Rat natural killer cell receptor systems and recognition of MHC class I molecules.

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

C1qRp elicits a Ca++ response in rat NK cells but does not influence NK-mediated cytotoxicity.

The cell surface receptor C1qRp (receptor for C1q, regulating phagocytosis) present on macrophages and neutrophils, is presumed to stimulate phagocytosis in these cells. However, C1qRp is also present on natural killer (NK) cells, and in these cells its physiological function is not currently known. We have investigated putative functions of this cell surface molecule in rat NK cells with the aid of two novel monoclonal antibodies (MoAb) LOV3 and LOV8 against rat C1qRp. NK cells are known to be potent cytotoxic effector cells, both through specific recognition of ligands on a target cell and killing of antibody-coated target cells (antibody-dependent cellular cytotoxicity, ADCC). NK cells prestimulated with MoAbs LOV3 or LOV8 did not exhibit altered ADCC. Furthermore, the addition of MoAb LOV3 or LOV8 to cytotoxic cultures of NK cells and Fc-receptor positive tumour cells did not affect killing in a redirected killing assay, indicating that the receptor did not influence NK cytotoxicity. However, this is the first paper to show that an intracellular Ca++-response is induced in rat NK cells upon stimulation of C1qRp with LOV3 and LOV8. The response induced by the antibodies was only minimally reduced in the presence of EGTA, indicating that most of the response is owing to the Ca++ mobilization from intracellular calcium stores.

Characterization and molecular cloning of rat C1qRp, a receptor on NK cells.

Here we report the generation of monoclonal antibodies (mAb) LOV3 and LOV8 to a 110-130-kDa membrane glycoprotein expressed by rat NK cells. This NK surface molecule was identified by eucaryotic expression cloning as the structural orthologue of the phagocytosis-stimulating receptor for complement factor C1q and mannose-binding lectin on human macrophages, C1qRp. Rat C1qRp is a monomeric type I integral membrane protein consisting of 643 amino acids with an N-terminal lectin-like domain, five epidermal growth factor-like domains, a transmembrane domain and a 45-residue cytoplasmic domain. It is encoded by a single gene on rat chromosome 3q41-q42 and is 67% and 87.5% identical at the amino acid level to human and mouse C1qRp, respectively. Rat C1qRp is expressed by resting and by activated NK cells, on subpopulations of NKR-P1(+) T cells (NK/T cells), dendritic cells, macrophages and granulocytes, but not by B cells or NKR-P1(-) T cells. Expression of this innate immune receptor is therefore not restricted to hematopoietic cells of the myeloid lineage, but is also expressed on subsets of cells of lymphoid origin. The mAb did not affect the cytotoxic function of NK cells, and C1qRp on NK cells may have functions not related to NK killing.

[Natural killer cells--important in the immunologic defense]. / Naturlige drepeceller--viktig immunologisk førstelinjeforsvar.

Natural Killer (NK) cells are a group of lymphocytes with a characteristic morphology and function. They are abundant in blood, spleen, liver and lungs. They are distinct from both T and B lymphocytes in their circulation patterns, profile of surface antigens, receptor repertoire and the way they discriminate between self and nonself. This latter NK function can partly be explained by an array of recently characterised NK receptors that can recognise and accurately discriminate between normal and altered MHC class I determinants. The basis for this discrimination is different from that of T cells and is discussed in this article. The role of NK cells in antimicrobial defense is well demonstrated, particularly that against viruses belonging to the herpesvirus group. A case report of a patient with a selective defect in NK cells and with recurrent viral infections is described. The role of NK cells in defense against malignant cells is more circumstantial, but NK cells do possess receptors which recognise tumour cells and kill them efficiently in vitro. A receptor which can recognise determinants unique for cancer cells has recently been described.

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.

Recruitment of alloreactive natural killer cells to the rat peritoneum by a transfected cell line secreting rat recombinant interleukin-2.

In order to obtain large numbers of natural killer (NK) cells from single rats for functional studies, we have devised a method for the generation of IL-2-activated NK cells in vivo. Rats were implanted intraperitoneally with cell-impermeable diffusion chambers (DC) containing cultures of transfected Chinese hamster ovary (CHO) cells secreting rat recombinant interleukin-2 (rIL-2). This resulted in a dramatic increase in the peritoneal exudate cell (PEC) number with a peak (300-1000 x 10(6)) 1 week after implantation. The majority were mononuclear cells of which a large proportion were CD3-NKR-P1+ NK cells, but with substantial numbers of macrophages (M phi) and CD3+8+NKR-P1+ T cells also. The NK activity against standard tumor target cells was high among PEC from six different inbred rat strains tested. However, the NK cell-mediated reactivity against concanavalin A (ConA)-activated T cell blasts from a panel of major histocompatibility complex (MHC) congenic strains differed widely. PEC from some strains (PVG, LOU/C, and AO) efficiently lysed all the MHC-disparate lymphoblasts. In other strains (BN and LEW) more restricted allorecognition repertoires were observed, whereas PEC from one strain (DA) were unresponsive. The secretion of rat rIL-2 intraperitoneally did not lead to a significant increase in the IL-2 level in the blood or in the total number or activity of NK cells in blood and spleen. The present method represents a most potent technique for generating large numbers of functional rat NK cells and shows the high efficiency with which IL-2 can induce NK cell recruitment in vivo.

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.