Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells

A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.     

Purified MHC class I molecules inhibit activated NK cells in a cell-free system in vitro

Natural killer cells have been shown to interact with MHC class I molecules via inhibitory receptors. However, it is not known whether the inhibition induced by MHC class I molecules requires other NK cell-target cell interactions. Thus, we examined whether purified MHC class I molecules alone were able to inhibit NK cell function. Purified H-2K(b) and H-2D(b) molecules inhibited the release of IFN-gamma from spleen (H-2(b))-derived lymphokine-activated killer (LAK) cell cultures stimulated by anti-NK1.1 antibody in a concentration-dependent manner. LAK cells generated from newborn mice that express low levels of MHC class I binding Ly49 inhibitory receptors were significantly less sensitive to inhibition by H-2K(b) compared to LAK cells from adult mice. Furthermore, LAK cells generated from spleen cells of Ly49C-transgenic mice were significantly more sensitive to inhibition by H-2K(b) compared to non-transgenic littermates. Taken together, the data indicate that MHC class I induced inhibition of NK cell mediated effector functions, as assessed by IFN-gamma release after NK1.1 triggering, does not require additional cell surface molecules other than MHC class I.     

Allogeneic versus syngeneic killer splenocytes as effector cells for the induction of graft-versus-tumor effect.

The effect of allogeneic versus syngeneic killer cells derived from normal or severe combined immunodeficiency disease (SCID) mice was evaluated for induction of antitumor reaction in a murine model of mammary carcinoma. Tumor cells of H-2d origin were injected intravenously into H-2(d/b) mice 24 hours after total body irradiation (4 Gy). On the following day, lymphokine-activated killer (LAK) splenocytes, derived from either minor (H-2d) or major (H-2b) histocompatibility complex (MHC)-mismatched parental normal mice or MHC (H-2b)-mismatched SCID mice, were given intravenously. LAK cells of H-2d normal or SCID mice, syngeneic to the tumor, were inoculated in parallel. The results show that LAK cells derived from minor histocompatibility complex-mismatched or MHC-mismatched parental normal mice improved the probability of tumor-free survival as compared with LAK cells syngeneic to the tumor cells, but they aggravated the severity of graft-versus-host disease. SCID splenocytes serving as a source of natural killer (NK) cells were expanded and activated in vitro by rIL-2 to obtain a sufficient number of DX5+ CD3- CD8- NK cells (SCID-LAK). H-2b SCID-LAK cells did not cause graft-versus-host disease and significantly delayed tumor growth compared with syngeneic H-2d SCID-LAK cells, as indicated by tumor colony assays in vitro and adoptive transfer experiments. However, the graft-versus-tumor effect was not long lasting, and treated mice finally died of tumor. Our results show an advantage of allogeneic over syngeneic cell therapy for achieving a graft-versus-tumor effect by rIL-2-activated T cells and NK cells. Periodic repetition of NK treatments may be required to achieve more durable antitumor effects.     

NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

Rejection of bone marrow cell allografts by natural killer cell subsets: 5E6+ cell specificity for Hh-1 determinant 2 shared by H-2d and H-2f.

The 5E6 antigen, defined by anti-5E6 mAb, is expressed on one-half of murine natural killer (NK) cells, and we have previously demonstrated (C. L. Sentman et al., J. Exp. Med. 1989. 170: 1991) that 5E6+ NK cells are necessary for the rejection of BALB/c (Hh-1d) but not C567BL/6 (Hh-1b) bone marrow cells (BMC). In experiments described here, we have characterized the specificity of 5E6+ and 5E6- NK cell subsets for hemopoietic histocompatibility-1 (Hh-1) antigens. Prospective recipient mice were treated with anti-5E6 mAb and challenged with BMC from a variety of donors. In addition, H-2d/Hh-1d C.B-17 scid 5E6+ or 5E6- NK cells were adoptively transferred into irradiated, NK cell-depleted hosts and challenged with H-2b/Hh-1b BMC. The data indicate that the 5E6+ NK cells are necessary for the rejection of only those BMC that express the Hh-1 determinant 2 shared by H-2d and H-2f haplotypes of strains BALB/c (d), A.Ca (f), and B10.M (f). No reactivity to other Hh-1 antigens resides in the 5E6+ population. In contrast, the ability of NK cells to lyse H-2d or H-2b tumor cells was independent of 5E6 expression. These results suggest that the 5E6 molecule is likely to be important in the specific recognition and rejection of BMC that express Hh-1 determinant 2, and is probably not involved in recognition of "tumor target cell structures".     

Induction, Specificity and Elimination of Asialo-GM 1+ Graft-Versus-Host Effector Cells of Donor Origin

In previous studies we demonstrated that an induced asialo-GM1 positive (ASGM1+) cell of donor origin that exerts natural killer cell-like activity (NK activity+) plays a crucial role in the development of graft-versus-host (GVH)-associated tissue damage and severe immunosuppression. This study examined whether the ASGM1+ (NK activity+) GVH effector cells were activated by non-specific signals or whether these cells were triggered by specific alloantigens and displayed antigenic specificity. C57B1/6 (B6) donor mice were treated with either B6 x AF1 (B6AF1) lymphoid cells and anti-asialo GM1 antibodies (anti-ASGM1) to induce and eliminate specifically activated B6-anti-B6AF1 ASGM1+ (NK activity+) cells or with polyinosinic: polycytidylic acid (poly I:C), and anti-ASGM1 to eliminate non-specifically activated ASGM1+ (NK activity+) cells. Donor spleen and lymph node cells depleted of the specific allo-induced ASGM1+ NK reactive cells showed near normal numbers of L3T4+ and Lyt-2+ cells and retained T- and B-cell functions as measured by mitogen responses (to PHA, Con A and LPS), mixed lymphocyte responses (MLR) (to B6AF1) and the generation of cytotoxic T cells (CTL) (to B6AF1 blasts). Anti-ASGM1 treatment almost completely abrogated NK activity in all donor inocula. GVH reactions were induced by injecting treated donor cells into B6AF1, B6 x C3HejF1 (B6C3HF1) and B6 x SJLF1 (B6SJLF1) hybrids and monitored by splenomegaly, suppression of T-cell mitogen responses and the development of histopathological lesions in the thymus, liver and pancreas. Cells from donors depleted of non-specifically (poly I:C) induced ASGM1+ cells induced severe histological lesions, marked immunosuppression and splenomegaly in all three F1 hybrid combinations. When the donor cells were depleted of specifically induced (B6-anti-B6AF1) ASGM1+ cells and injected into the three F1 combinations they induced splenomegaly in all three but caused severe tissue injury and intense immunosuppression only in B6C3HF1 and B6SJLF1 mice and not in B6AF1 mice. Genetic analysis suggests that the H-2D (or a closely related) region of the H-2 complex plays an important role in the activation of the specific GVH effector cells. These results suggest that the cell(s) responsible for splenomegaly are different from the ones that cause severe GVH-associated tissue damage and immunosuppression although there may be cells and/or lymphokines common to both processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype. Blocking Ly49-MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49-MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.     

Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.     

Selective elimination through a cytolytic mechanism of bovine serum albumin-specific T helper lymphocytes by T suppressor cells with the same antigen specificity

An antigen-specific T suppressor cell clone isolated from a CBA/J mouse tolerized to low doses of bovine serum albumin (BSA) has previously been analyzed with regard to its effector functions. The T suppressor cell clone HF1 specifically inhibits T helper cell responses to the antigen. It also has characteristic cytolytic activity which can neither be classified as cytotoxic T cell nor as natural killer cell activity. Since this lytic capacity might be of relevance in immunoregulation, it has now been studied in more detail. For that purpose BSA-specific T cell lines have been isolated from immune CBA/J mice in order to test them in 51Cr-release assays as possible targets for HF1 T suppressor cells. Two T cell lines, both BSA specific and restricted to recognition of I-Ek major histocompatibility complex determinants, have been selected for the studies because one is a helper cell (83/1), the other a suppressor cell type (83/2). HF1 T cells are able to lyse cells of line 83/1 but not those of line 83/2. Control experiments show that 83/1 cells are not a natural killer cell target and that on the other hand 83/2 cells are susceptible to lysis in an alloreactive BALB/c anti-CBA/J cytotoxic T cell response. The extent of lysis of 83/1 T cells by HF1 T cells changes with time after antigenic stimulation. The lysis is based on direct effector: target cell interaction and not caused by soluble mediators. The data are discussed with regard to the effector function of a type of T suppressor cells which expresses I-A and I-E molecules and whose proliferation is restricted to the recognition of I-A or I-E determinants.     

Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules

Natural killer (NK) cells are an essential component of the innate immunity toward tumors and virally infected cells. The function of NK cells is regulated by a precise balance between inhibitory and activating signals. These signals are mediated by NK cell receptors that bind either classical MHC class I molecules or their structural relatives such as MICA, ULBP, RAE-1, and H-60. Two separate families of NK cell receptors have been identified: the immunoglobulin-like family (KIR, LIR) and C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize the structure of Ly49 C-type lectin-like proteins hitherto solved (Ly49A, Ly49C and Ly49I) and their interaction with MHC class I molecules as determined by the co-crystal structure of Ly49A/H-2Dd and Ly49C/H-2Kb.     

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.     

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.     

Avian NK activities, cells and receptors

Natural killer (NK) activity has been examined in birds for over 30 years, but evidence that avian NK activity plays crucial roles in disease is only suggestive. In chickens, NK activity is mediated by TCR0 cells in the intestinal epithelium, but elsewhere subsets of αβ and γδ T cells (NKT cells) may be more important. There are few lectin-like NK receptor genes, located in the genomic region syntenic with the natural killer complex (NKC) as well as the major histocompatibility complex (MHC). In contrast, a huge number of Ig-like receptor genes are located in a region syntenic with the leukocyte receptor complex (LRC).     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

Tumor cell lysis by T cells distinct from NK cells and alloantigen-specific cytotoxic T cells

The generation and mechanism of tumor cell lysis by cytotoxic T cells derived from natural killer cell (NK) and allospecific cytotoxic T cell (CTL)-depleted precursors were examined. NK cells and the precursors of alloantigen-specific CTL were deleted from human peripheral blood lymphocytes by preincubation with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Following phytohemagglutinin activation, CD3(+), CD4(+) or CD8(+), CD11b(-), CD16(-), and NKH1(-) killer cells capable of lysing a broad spectrum of tumor targets were generated. Cytolysis was not strictly lectin dependent as similar killer cells were generated by activating Leu-Leu-OMe-treated T cells with immobilized monoclonal antibodies to the CD3 molecular complex. The rate of tumor cell lysis by these mitogen-activated T cells was slower than that mediated by CD3(-) NK cells. Tumor cell lysis by mitogen-activated killers was inhibited by anti-CD3 but was not restricted by major histocompatibility complex antigen expression on target cells or by CD4/CD8 expression on effectors. Although similar to NK cells in susceptibility to anti-LFA-1 inhibition of killing, these mitogen-activated killer cells were more sensitive to the inhibitory effects of anti-CD2 than were CD3(-)-activated NK-like cells. Thus, tumor cell lysis by CD3(+) cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes appears to be mediated in part by mechanisms distinct from those employed by CD3(-) NK cells or antigen-specific CTL.     

Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen

The study of natural killer (NK) has been difficult because they account for a small percentage of peripheral blood and splenic lymphocytes and the paucity of NK specific antigens that have been identified. We have isolated pure populations of C57BL/6 (H-2b) NK cells using the IgG2b monoclonal antibody PK136 (anti-NK1.1). These NK1.1+ cells were used to immunize 129/J (H-2b) mice, and in this report, we describe three new NK specific monoclonal antibodies (SW3A4(IgM), SW4B12(IgG1), and SW2B4(IgG2b] and their relationship to the known murine NK antigen NK1.1. We have further characterized the NK1.1 antigen as a 39 kd molecule which is coded for by a gene which appears to map to chromosome 6.     

Absence of major histocompatibility complex class I on neural stem cells does not permit natural killer cell killing and prevents recognition by alloreactive cytotoxic T lymphocytes in vitro

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2(b)) mice were explanted and cultured to expand NSCs. Analysis of P2-P17-cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN gamma) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN gamma NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN gamma for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN gamma-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2(d)) allospecific anti-H-2(b) CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.     

Generation of cellular immunity to lymphocytic choriomeningitis virus is independent of CD1d1 expression.

CD1 molecules are cell surface glycoproteins, structurally similar to major histocompatibility complex (MHC) class I molecules. The murine CD1d1 molecule has been shown to be essential for the positive selection of a unique subpopulation of T cells [the natural killer (NK) T cells], as CD1d1-deficient mice lack NK T cells. These cells have recently been suggested to play an important role in the induction of innate immunity (i.e. NK cells) and the regulation of immune homeostasis. As such, it was asked whether NK T cells were necessary for the generation of cellular immunity to an acute virus infection. In these studies, the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), a classic inducer of NK cells, and its pathogenic variant clone 13 were used. When NK-cell activity was assessed on day 3 post-LCMV infection, surprisingly, it was found that CD1d1-deficient mice could generate NK-cell activity at wild-type levels. Likewise, LCMV-specific cytotoxic T-lymphocyte (CTL) activity in CD1d1-deficient mice was indistinguishable from that generated in wild-type mice. Additionally, viral titres in the spleen (LCMV Armstrong) and blood (LCMV clone 13) of infected CD1d1-deficient mice were at comparable levels to those found in wild-type mice, as were virus infection-induced increases in cell surface H-2Kb in the spleen. Therefore, these results suggest that the LCMV-induced generation of NK-cell and virus-specific CTL activity, as well as viral clearance, are independent of CD1d1 expression.     

A potent inhibitor of protein kinase C inhibits natural killer activity

A potent inhibitor of protein kinase C(PKC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), dose-dependently inhibited natural killer (NK) activity in large granular lymphocytes (LGL) pretreated at 37 degrees C for 30 min. However, neither N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9), which inhibits more effectively cyclic nucleotide-dependent protein kinases than other kinases, nor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), which was used as a control for H-7, reduced NK activity. The inhibitor effect of H-7 was not due to changes in effector cell viability or target cell binding. We also found that H-7 suppresses PKC activity in both the cytosol and membrane fractions of LGL. From these findings, PKC is considered to play an essential role in the lytic mechanism of NK cell-mediated cytolysis.