Enhancement by interferon of chicken splenocyte natural killer cell activity against Marek's disease tumor cells

A non-immune natural killer-type cell population (NK) from 6-to 12-week old chickens was able to kill MSB-1 Marek's disease (MD) tumor cells in vitro; as measured by the 51Cr-release cytotoxicity assay. Removal of T cells, B cells, adherent cells, or any combination of the three populations of cells did not result in diminished levels of cytotoxicity of the remaining spleen cells against MSB-1 cells. The cytotoxicity of chicken NK cells could be rapidly augmented by polyinosinic-polycytidylic acid (poly I:C) and by the Cal 11914 strain of Newcastle disease virus (NDV), but not by the TCND strain of NDV which is not an interferon (IFN) inducer, indicating that IFN play a role in augmentation of the NK activity in chickens.     

Existence of cytotoxic activity against BLV-transformed cells in lymphocytes from normal cattle and sheep

Peripheral blood lymphocytes (PBL) from normal cattle and sheep were tested for their cytotoxic activity against several target cells using a 20-hour 51Cr release assay. The following characteristics of the effector cells were observed; 1) PBL from animals showed cytotoxic activity against two sheep cell lines (FLK and SF-28) that were transformed with bovine leukemia virus. However, normal sheep and bovine cells and Molony leukemia virus-induced mouse lymphoma cell line (YAC-1) were not killed by these cells. 2) A time course study showed that the activity was first observed at 4 to 8 hours and reached a maximum at 20 to 30 hours after incubation. 3) Cytotoxic activity was observed in both adherent and nonadherent cell fractions when PBL were passed through a nylon-wool column. This indicated that the effector cells showed some degree of adherence. 4) Treatment of PBL with carrageenan did not change the cytotoxic activity against target cells, indicating that phagocytic capability is not perhaps necessary for cytotoxicity to take place. These results indicate that the effector cells participating in the cytotoxic reaction resembled natural killer cells or natural cytotoxic cells which are present in murine and human systems. However, analysis of the cell surface markers of the effector cells is yet to be done in future studies.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

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.     

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.     

Therapeutic effects of Toxoplasma lysate antigen on 20-methylcholanthrene-induced BALB/c mouse tumors.

Therapeutic effects of Toxoplasma lysate antigen (TLA) were studied in mice bearing the tumor in the second passage of 20-methylcholanthrene (MC)-induced tumor cells. Intramuscular administration of TLA 7 days after the tumor-cell inoculation caused apparent inhibition of the tumor growth on day 14. The second treatment facilitated the therapeutic effects. Intravenous transfer of spleen cells prepared from TLA-sensitized mice into tumor-bearing mice also represented the growth inhibitory effects. Prominent effects were seen when the transferred cells were prepared 5 days after sensitization of donor animals. The inhibitory effects were absent in the groups transferred only the adherent cells or the non-adherent cells prepared from sensitized mice. The strongest inhibitory effect was observed in the group to which both adherent and non-adherent spleen cells were transferred simultaneously from sensitized mice. In in vitro experiments, spleen cells obtained from sensitized mice showed cytolytic effect on P-815 or YAC-1 cells after the secondary stimulation in vitro with TLA. Large non-adherent cells containing densely packed granules were induced when cultured with the adherent cells obtained from sensitized mice. These results revealed that TLA can inhibit the growth of the chemically-induced transplantable tumors by activation of adherent and non-adherent spleen cells.     

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.     

Cell-mediated cytotoxicity of bovine mononuclear cells to IBRV-infected cells: dependence on Sephadex G-10 adherent cells

Following intranasal inoculation of cattle with infectious bovine rhinotracheitis virus (IBRV) mononuclear cells that produced a genetically unrestricted cytotoxic response against IBRV-infected, but not against uninfected cells, were present in peripheral blood. Cytotoxicity was detected between 6 and 14 days after primary infection in a 20 h, but not in a 5 h, 51Cr-release assay. Cytotoxic activity was present in peripheral blood mononuclear cells from infected and subsequently hyperimmunized cattle for a considerably longer time. Neither natural cytotoxicity, antibody-dependent cell cytotoxicity, nor antibody produced during the assay was responsible for the cytotoxicity. However, cytotoxicity was dependent upon an adherent mononuclear cell that was partially removed by passage over nylon wool and completely removed by passage over Sephadex G-10.     

Characteristics of cytotoxic cells induced by Toxoplasma lysate antigen in mouse spleen.

Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL male-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen cells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive, and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK) cells induced by IL-2.     

Natural immunity against ovarian tumors

We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modified tumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NK-resistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Comparative viral, immunologic, and pathologic responses of chickens inoculated with herpesvirus of turkeys as embryos or at hatch

Chickens were vaccinated with the herpesvirus of turkeys (HVT) at embryonation day 17 or 18 or at hatch and various responses of the 2 vaccinated groups were compared. In embryo-vaccinated chickens, HVT titers were high in the lungs before HVT could be isolated from other tissues. Seemingly, embryos acquired infection via the respiratory tract. In hatch-vaccinated chickens, HVT was first isolated from spleen and then from other tissues. Titers of recoverable HVT in tissues of embryo-vaccinated chickens were higher than in those of hatch-vaccinated chickens, particularly during the 1st week of age. Anti-HVT antibodies and natural killer cell reactivity in spleen effector cells were comparably increased in both vaccinated groups. Embryo vaccination with HVT did not cause progressive lesions, reduction in body weight gain, or impairment of humoral and cellular immune functions. Seemingly, HVT can be used safely as an embryonal vaccine in chickens.     

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the (51)Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to (51)Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.     

Immunization against Marek's disease transplantable cell lines

Continuous passage of MDCC-RP1, a highly tumorigenic Marek's disease (MD) lymphoblastoid cell line, in cell culture resulted in a gradual loss in ability of the cell line to cause progressive tumors in susceptible day-old chicks. Inoculation of day-old chicks with high-cell-culture-passaged (187th to 417th) nontumorigenic MDCC-RP1 cells gave excellent protection against challenge at 8 days with low-passaged tumorigenic MDCC-RP1 cells but failed to protect against primary tumors caused by inoculation with MD virus. Vaccination with the herpesvirus of turkeys, on the other hand, protected the chickens well against primary tumors caused by MD virus and against transplantable tumors caused by tumorigenic MDCC-RP1 cells, but it did not protect as well against another MD lymphoblastoid cell line, MDCC-RP4. It is unlikely, therefore, that vaccines prepared from passaged MDCC-RP1 cell lines will have value for protecting chickens against MD in the field.     

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.     

重组鸡白细胞介素18(rChIL-18)对MDV感染SPF鸡细胞免疫的影响

用马立克氏病病毒（MDV）感染5日龄SPF鸡后,取21日龄和35日龄鸡的淋巴细胞,运用3H-TdR掺入法检测T淋巴细胞对ConA和重组鸡白细胞介素18（rChIL-18）的反应,并用MTT法检测NK细胞和CTL对MD肿瘤细胞系CU147的杀伤活性及rChIL-18和IFN-γ对它们的作用。结果显示,SPF鸡感染MDV后淋转水平显著下降,NK细胞、CTL杀伤活性在感染后21日龄时升高,而在35日龄时NK细胞杀伤活性显著下降。rChIL-18对对照组和感染组SPF鸡的淋转水平和杀伤活性均有提高作用,同样IFN-γ也具有提高NK细胞和CTL杀伤活性的作用。     

Comparative pathogenesis studies with oncogenic and nononcogenic Marek's disease viruses and turkey herpesvirus.

An apparently nononcogenic Marek's disease virus (SB-1) and turkey herpesvirus could be readily isolated from spleen, bursa of Fabricius, thymus, and peripheral blood lymphocytes of chickens beginning 4 to 6 days after inoculation, but unlike infections with two isolates of oncogenic Marek's disease virus (JM-10 and CU-2), virus replication in these cells was rare, and necrosis in the organs was essentially absent. Splenic enlargement was observed regularly during the first 4 to 11 days after inoculation, and Marek's disease tumor-associated surface antigen was observed on splenic and other lymphocytes in the four viral inoculation groups. Cellular cytotoxicity of splenic lymphocytes was demonstrated in vitro with cultured Marek's disease tumor cells (MSB-1 lymphoblastoid cell line) as the target in a chromium-release assay. The four viral infections induced sensitized lymphocytes.     

Serial transfer of a transplantable tumor: implications for Marek's vaccine mechanisms

The mechanism of Marek's disease (MD) vaccination to prevent the lymphoproliferative disease in chickens is not well understood. It is generally recognized that vaccination prevents disease, including the induction of T-cell tumors, but it does not prevent the pathogenic virus from infecting and replicating in the vaccinated host, nor does it prevent bird to bird spread of the oncogenic virus. The stage at which the vaccinated immune system intervenes in the process from infection to the induction of tumors remains obscure. Using a transplantable tumor induced by the Md5 strain of MD virus (MDV), we show that CVI988 vaccination does not prevent the induction of transplantable tumors in the 15I(5) x 7(1) chicken line. A monoclonal tumor with a V beta 1 T-cell receptor spectratype of 207 base pairs was used to follow the transplantable tumor in serial passages in vivo. This transplantable tumor could be passed in vaccinated birds. The length of time between vaccination and challenge (5 to 12 days) had little or no influence on the ability to transfer the tumor. There was variability in the manifestation of the disease produced by the transplanted tumor. Some chickens presented as normal but were still capable of transmitting the transplanted tumor to newly vaccinated recipients via their blood. This indicates that some chickens can control, but not eliminate, the tumor. The variables inducing health or disease in the challenged chickens remain obscure, but environmental or other factors likely depress the immune system allowing the tumor to overwhelm the immune system.     

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells     

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio)

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.