Pyrimidinones: inducers of NK cell activity and antitumor immunity

We have shown that pyrimidinone molecule, 2 amino-5-iodo-6-phenyl-4-pyrimidinone (AIPP) after regional administration, displayed both prophylactic and therapeutic effect on ascitic mammary adenocarcinoma, ACA-755. The antitumor effect was mediated by natural killer (NK) cells, since depletion of this lymphocyte population resulted in abrogation of AIPP-induced tumor resistance. This observation confirms the role of NK cells in antitumor immunity and suggests that AIPP may be therapeutically beneficial also in man.     

Natural killer (NK) activity of porcine blood lymphocytes against allogeneic melanoma target cells

Allogeneic PM/86 melanoma cells of Munich Troll miniature swine have been used for the demonstration of porcine peripheral blood NK cell activity. Compared with the specific lysis of xenogeneic K562-, U937- and Vero-target cells, NK cell-mediated cytotoxicity (NK-CMC) against PM/86 melanoma tumor cells was significantly lower in a 16 h chromium release assay. The target cell susceptibility to peripheral blood NK-CMC of both adult Troll miniature swine and German Landrace sows was very similar. Cold target inhibition assays revealed the allogeneic PM/86 melanoma cells to be the most powerful inhibitors of NK-CMC. Nylon wool non-adherent lymphocytes produced interferon (IFN)-alpha in different quantities upon contact with NK susceptible target cells. The NK effector cells could be stimulated to a higher lytic activity against all susceptible targets by a moderate dose of natural human interleukin-2 (nhuIL-2). The role of NK-CMC in melanoma tumor rejection and/or prevention of metastases is yet unknown in swine although porcine melanoma serves as a good model for the disease in man.     

Interleukin-2 corrects defective NK activity of patients with leukemia

NK cells of patients with leukemia display low cytotoxic potential. Since the NK cells have been suggested to play a role in natural resistance to leukemia, we considered it of importance to investigate the approaches leading to the correction of NK defect of leukemic patients. Our studies demonstrate that culture of effector cells with interleukin-2 (IL-2) resulted in restoration of cytotoxic defect. This was indicated by normalization of tumor-binding as well as lytic NK activity, by normal frequency of cytotoxic cells and their ability to recycle. The NK cell nature of cytotoxic cells was shown by abrogation or depletion of cytotoxicity by antibody directed against NK cell-associated, but not T cell-associated antigen. The generation of NK cell activity against fresh leukemic cells suggests that adoptive transfer of IL-2 activated NK cells may be a new approach to leukemic treatment.     

Cytotoxicity of bovine lymphocytes after treatment with lymphokines

Cytotoxic lymphocytes were generated from bovine peripheral blood mononuclear leukocytes after in vitro stimulation with lymphokines that contained interleukin-2. Lymphokine-stimulated cultures were cytotoxic to K562 cells (human natural killer [NK] targets) and YAC-1 cells (mouse NK targets), but not to HSB-2 cells (human NK targets) in a 4-hour, 51Cr-release assay. Cells generated after lymphokine activation also mediated antibody-dependent cellular cytotoxicity to HSB-2 cells. Appearance of effector cells as a function of time in culture, method of stimulation, and cold target competition experiments strongly indicated that direct cytotoxicity and antibody-dependent cellular cytotoxicity may have been mediated by the same cell. Cells generated by similar conditions were able to mediate cytotoxicity against infectious bovine rhinotracheitis virus-infected target cells, especially in an 18-hour assay.     

Flow cytometric analysis of chicken NK activity and its use on the effect of restraint stress

Immune system is organized by the influence of both neural and endocrine systems. NK activity plays an important role in the innate immunity. In this study, we observed the effects of restraint stress on chicken peripheral blood NK activity. Viability of FITC-labeled RP9 was measured with PI after treatment with the effector cells. Chicken peripheral blood CD8alpha+ cells expressed strong cytotoxic activity, in contrast to thrombocytes, while peripheral blood CD3+ CD8alpha+ cells and CD4+ cells had little cytotoxic activity. Con A supernatant enhanced the cytotoxic activity of CD8alpha+ cells. Therefore, it is considered that these cytotoxic activities measured by flow cytometry (FCM) analysis are NK activity. When chickens were exposed to restraint stress, the levels of serum corticosterone increased transiently over a short period of time while the NK activity decreased. The decreased NK activity, however, did not recover to the intact levels for a long time, even once the serum corticosterone levels had recovered. These data indicate that chicken NK activity is able to be measured by flow cytometric analysis and that restraint stress causes severe damage to the chicken NK activity.     

Tumoricidal effect of interleukin-2-activated killer cells in canines

We investigated the effect of both partially purified (TCGF) and recombinant interleukin-2 (rIL-2) on the tumor-directed cytotoxic activity of canine peripheral blood mononuclear cells (PBMC) using three normal canines. No cytotoxic activity was displayed by unstimulated effector cells at 3 h of incubation; however, the cytotoxic effect was observed in a 16-h assay. PBMC of all canines displayed significant levels of lytic activity after stimulation for 4 to 7 days with both types of IL-2 against a variety of allogeneic and xenogeneic neoplastic cells in 3-h 51Cr release assay. The cytotoxic activity of cultured cells increased proportionally in the 16-h assays. Morphological examination of the May-Grünwald and Giemsa stained cytocentrifuged slides of cultured cells on each day of assay showed an increase in large granular lymphocytes (LGLs) beginning on day 4 and reaching a peak on day 7 of culture.     

Presence of adherent cytotoxic cells and non-adherent natural killer cells in progressive and regressive Marek's disease tumors

Marek's disease virus-induced progressive tumors and Marek's disease transplantable local regressive tumors were dispersed by treatment with collagenase and cells were examined in vitro for cytotoxic effector functions against target cells of tumor lines. Both types of tumors had adherent and non-adherent cytotoxic cells. The cytotoxicity of adherent cells was detected in an 18-hour but not in a 4-hour 51Cr-release assay. The adherent effector cells from progressive tumors were inactivated by pretreatment with carbonyl iron and carrageenan whereas the adherent effector cells from the regressive tumors were refractory to these treatments. In the progressive tumors, the 18-hour cytotoxic activity of cells of tumors and of spleens of tumor-bearing chickens was compared; the activity was higher in the tumor than in spleen. The nonadherent cell cytotoxicity detectable in a 4-hour 51Cr-release assay was associated with anti thymocyte serum-resistant natural killer cells. The incidence and levels of natural killer cell reactivity were greater in the regressive tumors than in the progressive tumors. In the regressive tumors, the natural cytotoxicity levels were higher in the tumor than in the spleen of tumor-bearing chickens. The differences in characteristics of adherent cytotoxic cells in virus-induced progressive tumors and transplantable regressive tumors and elevated levels of NK cells in regressive but not in progressive tumors may indicate a role for intratumoral immunity in tumor regression.     

The immune reactivity of the regional and distant lymph nodes to autochthonous ovine squamous cell carcinomata

Anti-tumor immune reactivity of lymphocytes derived from lymph nodes regional to and distant from tumor growth, as well as that of peripheral blood leucocytes, against autochthonous tumor cells, was investigated. Experiments were carried out in vitro using a 51Cr cytotoxic assay and in vivo by cannulating the afferent and efferent lymphatics of regional and distant lymph nodes and challenging via the afferent lymphatics with 10(7) cultivated autochthonous tumor cells. No anti-tumor cytotoxic reactivity was detected in vitro using lymphocytes derived from any of the sources studied. In vivo, while challenge with autochthonous tumor cells produced no response in the regional lymph node, significant blast cell response was obtained in the distant node. The response at the distant node was associated with the production of antibodies that could bind to tumor cells without causing their demise. The anergy observed at the regional lymph node, and the possibility of a relation between the events occurring at that node and those observed at the distant node, are discussed.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Natural killer cell activity of chicken intraepithelial leukocytes against rotavirus-infected target cells

Intraepithelial leukocytes (IEL) and splenocytes collected from uninfected and rotavirus-infected chickens were evaluated for cytotoxic activity against a natural killer (NK) cell-susceptible lymphoblastoid cell line (LSCC-RP9) and against rotavirus-infected chick kidney cells in 4-h chromium-release assays. Both splenocytes and IELs from uninfected and rotavirus-infected chickens were cytotoxic for LSCC-RP9, and the levels of this NK cell activity were not altered by infection of the host with rotavirus. IELs but not splenocytes from uninfected and rotavirus-infected chickens were cytotoxic for rotavirus-infected but not for uninfected chick kidney cell targets. Because this cytotoxic activity was not induced nor altered by rotavirus infection of the host, and was not major histocompatibility complex-restricted, it was considered to be due to NK cell activity. The cytotoxicity of IELs against rotavirus-infected target cells was dose-dependent; however, there was some suppression of cytotoxic activity at high effector to target cell ratios. There were no differences in the cytotoxic activities of IELs collected from the duodenum versus the jejunum. The in vitro cytotoxic activity of IELs against rotavirus-infected target cells suggested that NK cell activity may be an important immune response to rotavirus infections in vivo. The absence of cytotoxic activity by splenocytes against rotavirus-infected target cells indicated that there may be different subpopulations of NK cells in the spleen and intestinal epithelium of chickens.     

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8⁺ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8⁺ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3⁺, CD4^?, CD21^?, CD5^lo, α/βTCR⁺, and γ/δTCR^? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8^? cells. This subset of CD8⁺ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8^? cells. In contrast, CD8⁺ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8⁺ cells are an important subset associated with NK cytotoxic activity.     

Natural cell-mediated cytotoxicity to cells infected with infectious bovine rhinotracheitis virus

Cell-mediated cytotoxicity against viral-infected cells was demonstrated in a 6-hr 51Cr release assay. Peripheral blood mononuclear leukocytes from both infectious bovine rhinotracheitis virus (IBRV)-infected and noninfected cattle exhibited preferential lysis against IBRV-infected primary bovine embryonic kidney (BEK) cells compared to cells infected with pseudorabies virus and noninfected BEK cells. Addition of specific antibody to the assay did not enhance cytotoxicity. The effector cell was a nonadherent cell which was either spontaneously enriched or generated during in vitro cultivation. Maximal cytotoxic activity was detected in peripheral blood mononuclear cells cultured for 3 to 5 days. Several factors affected the magnitude of cytotoxicity during the assay: target cell type, concentration of viral inoculum, duration of effector and target cell contact. It is suggested that target cell lysis was a form of natural cell-mediated cytotoxicity mediated by a cell which has different characteristics from the typical human and murine NK cell.     

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells.

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.     

Characteristics of Yorkshire swine natural killer cells

The present study examined the properties of NK activity in Yorkshire swine. The results support other porcine studies which indicate the swine NK system has both similarities and differences to this system in other species. Profiles of NK activity indicated swine NK cells are highly reactive against the YAC-1 lymphoma, the K-562 myeloid leukemia, the P-815 mastocytoma, and the TU-5 virally transformed fibroblast. In contrast, the MOLT-4 and SB leukemias are NK resistant. Kinetic studies indicated that in Yorkshire swine, NK lysis begins 6 h after mixing effectors and targets. The kinetics of the lytic reaction differ both from other breeds of swine and from other species, where cytotoxicity is readily measured in 4-h assays. The delayed lysis was not due to delayed target cell recognition, because Yorkshire swine NK cells are rapidly bound to tumor targets. The delayed lysis seems to be due to a refractoriness in the NK lytic mechanism. This delay may relate to the morphologic finding that the target-binding cell in Yorkshire swine appeared quite different from the large granular lymphocyte (LGL) reported as the NK effector in humans and rodents. Indeed, in light microscopic studies the typical tumor-binding cell in Yorkshire swine is a small, apparently nongranular, lymphocyte. Analysis of NK activity at the single cell level was performed with single effector-tumor conjugates immobilized in agarose. Generally, lysis by target binders paralleled sensitivity to lysis in 51Cr release tests, indicating lysis in agarose may be used as an NK index in swine. Like other species, swine NK cells were found to be nonadherent lymphocytes with a characteristic tissue distribution. Peripheral blood and spleen had the highest levels of NK activity. Lymph node cells displayed a small amount of NK activity which was limited to the YAC-1 target, while thymocytes showed no appreciable NK activity against any of the cell lines tested.     

Monoclonal precipitating antibodies to porcine immunoglobulin M

Fusion of splenic immunocytes from a porcine IgM-immunized BALB/c mouse with SP2/0 mouse myeloma cells resulted in 231 primary hybrids. Culture fluids of the primary hybrids were screened for antibody production by enzyme-linked immunosorbent assay (ELISA) against porcine IgM and by radial immunodiffusion versus porcine serum. Culture fluids of 10 of the primary hybrids were positive in IgM-ELISA and radial immunodiffusion. Six of these primary hybrids (1A11, 1D10, 2D7, 2E2, 3B11, and 5C9) were cloned, and ascitic fluids were produced using cloned primary hybrids. The monoclonal antibodies (Mabs) in ascitic fluids were characterized as to their reactivity with porcine immunoglobulin isotypes. All six Mabs had mouse IgG1, K isotype and were mu-chain specific as they formed single precipitin lines against porcine serum and porcine IgM and no lines against porcine IgG, IgA, and fetal porcine serum in immunodiffusion and immunoelectrophoresis. In indirect ELISA, all Mabs reacted with porcine serum, porcine IgM, and mu-chains but did not react with porcine IgG, IgA, or light chains. All six Mabs were species-specific and recognized either of two antigenic regions of mu-chain. These Mabs have been successfully used to detect IgM-containing cells in tissue sections, to detect IgM in serum, and to quantitate surface membrane IgM-bearing cells in peripheral blood.     

Characterization of porcine xenoreactive cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatibility complex.     

Cell mediated immune responses in ponies following infection with equine influenza virus (H3N8): the influence of induction culture conditions on the properties of cytotoxic effector cells

Cytotoxic cell precursors and/or cytotoxic memory cells were demonstrated in the peripheral blood of ponies after aerosol infection with influenza A/equine/Newmarket/79 (H3N8). In order to reveal their cytotoxic potential, peripheral blood mononuclear cells required a secondary antigenic stimulation. In vitro induced cytotoxic cells showed activity against influenza infected target cells in a 3-4 h 51Cr-release assay. The reactivity of cytotoxic cells was markedly influenced by the conditions of the secondary induction culture. If high concentrations of exogenous crude equine IL-2 were used, virus infected target cells were susceptible to lysis by autologous or allogeneic effector cells. However, if IL-2 concentration was reduced, cytotoxic cells were generated which showed features consistent with cytotoxic T cells in that target-cell killing was genetically restricted.     

Host environment as a modulating factor of swine natural killer cell activity

The large granular lymphocyte (LGL) population includes such heterologous effector cells as the natural killer (NK), lymphokine activated killer (LAK), antibody dependent cellular cytotoxic (ADCC) and non-MHC restricted T cells. These LGL subpopulations have all been associated with NK activity. In some species, enhanced NK activity is correlated with exposure to viral, bacterial and parasitic agents. Consequently, the host environment could serve as a modulatory factor of NK activity in laboratory animals. During our investigation of tumor regression in melanoma swine, we observed marked differences in the NK activity of peripheral blood lymphocytes collected from two separate groups of Sinclair melanoma miniature swine maintained under different conditions. Group A pigs were vaccinated and extensively treated for endo- and ectoparasites while group B swine were not. In addition, chronic exposure to infectious and parasitic diseases have been documented in the group B swine. Peripheral blood NK activity was assessed by standard in vitro 4-h chromium release assays. The NK activity of group B swine was markedly exaggerated when compared to group A swine. Thus, the significance of NK activity may be distorted as a result of the modulating effect of pathogen exposure.     

NKp46 defines ovine cells that have characteristics corresponding to NK cells

ABSTRACT: Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.