Cell-mediated cytotoxicity of lymphocytes from chickens inoculated with herpesvirus of turkey against a Marek's disease lymphoma cell line (MSB-1).

Using a new device which increases the sensitivity of detection of specific immune lysis of target cells by labeling them with [35S]-methionine, the in vitro cell-mediated cytotoxic response of spleen lymphocytes and peripheral blood lymphocytes from chickens vaccinated with herpesvirus of turkey (HVT), O1 strain, against MSB-1 line cells was clearly demonstrated. The cytotoxic activity was clearly inhibited by pretreatment of effector lymphocytes with anti-T lymphocyte serum and complement. The activity was greater using T cells purified from spleen lymphocytes and peripheral blood lymphocytes than with the unfractionated cells, indicating that T lymphocytes play the main role in effector activity. Using sera from HVG-vaccinated chickens, no significant cytotoxic effects were detected in the complement-dependent antibody cytotoxicity test against MSB-1 cells. These results suggest that cellular immunity against the surface antigen of Marek's disease (MD) lymphoma cells is mainly related to the preventive mechanism against MD incidence by HVT vaccination.     

Interactions in vitro between sporozoites of Eimeria tenella and host peritoneal exudate cells: electron microscopal observations

The interactions of sporozoites of Eimeria tenella with peritoneal cells from normal and from immunized chickens were examined in vitro. Although the uptake of sporozoites by cells from immunized birds was greater than by cells from susceptible birds, no differences were apparent in their appearance in electron micrographs. Entry into both macrophages and heterophils (comparable to mammalian neutrophils) was by phagocytosis. The findings are discussed.     

Monoclonal antibodies reactive with chicken peritoneal macrophages: identification of macrophage heterogeneity

Mouse monoclonal antibodies (MCAs) were prepared against chicken inflammatory macrophages for the purpose of analyzing macrophage heterogeneity. Macrophage-rich adherent peritoneal exudate cells harvested from Cornell K-strain chickens 42 hr after stimulation with Sephadex were used as immunogens in the production of the monoclonal antibodies. Eight hybridoma clones producing antibodies reactive with chicken peritoneal macrophages were subjected to characterization. While six of the monoclonal antibodies cross-reacted with various hematopoietic cell types, two MCAs (CMTD-1 and -2) were restricted in reactivity to macrophages. CMTD-1 was found to react with activated peritoneal macrophages generated by several irritants. In contrast, CMTD-2 identified a subpopulation of macrophages elicited by specific carbohydrate-based stimulants. This presumably was based on the cross-reaction of this MCA with specific carbohydrate linkages. Analysis using flow cytometry revealed the time-dependent appearance of CMTD-2-positive peritoneal macrophages between 24 and 52 hr after ip Sephadex injection. This subpopulation of peritoneal macrophages was found to be heterogeneous for the ability to undergo in vitro phagocytosis of sheep erythrocytes. CMTD-2-positive cells were also detected in the thyroids of 2-week-old Obese strain chickens with spontaneous autoimmune thyroiditis and at a low incidence in the spleens of normal chickens.     

Suppressive Effect by Cooperation of Splenic and Peritoneal Adherent Cells on Generation of Cytotoxic T Lymphocytes

When spleen cells primed in vivo against allogeneic lymphoid cells were used as responder cells in secondary mixed lymphocyte cultures, a high degree of cytotoxicity was generated even in the absence of splenic adherent cells. However, removal of adherent cells from such primed responder spleen cells reduced the cytotoxicity to some extent. On the other hand, when these responder cells were transferred into the peritoneal cavity of irradiated syngeneic mice together with antigenic cells, unseparated responder cells generated a lower degree of cytotoxicity than did adherent cell-depleted responder cells. In an in vitro system, peritoneal adherent cells also suppressed the generation of cytotoxic T lymphocytes by unseparated responders; however they augmented the cytotoxic T lymphocyte generation by adherent cell-depleted responders. These adherent cell populations with augmenting activity became inhibitory when they coexisted. The mechanism of this inhibitory action remains unclear.     

Neonatal susceptibility to MHV3 infection in mice. I. Transfer of resistance.

Up to 3 weeks of age, mice of the resistant A/J strain are fully susceptible to mouse hepatitis virus type 3 infection (MHV3). Immune deficiency, however, resulting from neonatal thymectomy or long term ALS administration led A/J animals to remain susceptible when tested at adult age. Whole spleen cells transferred from normal adult A/J donor mice protected suckling syngeneic recipients from i.p. infection with MHV3. Such a protective capacity of spleen cells was abolished after treatment with anti-theta serum and complement. Spleen cell separation by means of adherence to plastic also showed that neither the nonadherent nor the adherent populations injected separately were able to confer resistance to young mice challenged with the virus. Protection was not achieved with peritoneal cells originating from adult syngeneic animals. Transfer of resistance to MHV3 was obtained, however, when peritoneal cells were associated with adherent spleen cells. This study indicated that two types of mature cells, at least, were required for transferring MHV3 resistance into newborn mice of the A/J strain: T lymphocytes and an adherent spleen cell population.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

Mechanisms of T cell hyperreactivity in obese strain (OS) chickens with spontaneous autoimmune thyroiditis: lack in nonspecific suppression is due to a primary adherent cell defect

The disturbed homeostasis of the immune system in Obese strain (OS) chickens with spontaneous autoimmune thyroiditis consists in a general T cell hyperreactivity (concanavalin A hyperresponsiveness, interleukin 2 (IL 2) hypersecretion), particularly expressed by those lymphocytes which infiltrate the diseased thyroid gland. This abnormality has been attributed to a defective regulation of both IL 2 production and IL 2 function by low m.w. factors, which are present in serum and splenocyte culture supernatants of normal chickens, but deficient in the OS. In the present study we identified the cellular origin of IL 2 antagonistic activity as a nonlymphoid, adherent cell. Suppressor factor production in vitro was confined to the plastic adherent fraction of spleen cells and preincubation of splenocytes with nylon wool, silica particles, or carbonyl iron significantly reduced the nonspecific suppressive activity of the culture supernatant. Kinetic studies revealed the defect in nonspecific suppression to entail prolonged IL 2 production by concanavalin A-activated OS spleen cells. In vivo treatment of normal White Leghorn chickens with silica led to a decrease in suppressive serum activity down to the OS level, whereas neither neonatal thymectomy nor bursectomy had any effect. The defective suppressor factor production in autoimmune chickens appeared to be due to a functional, but not numeric defect of macrophages as revealed by phenol red staining. The possibility that this aberration in adherent cell function might be a secondary phenomenon to the recently described reduced corticosterone tonus in OS chickens was excluded by in vivo substitution with exogenous glucocorticoids which did not normalize the suppressor defect in serum or in conditioned medium. Finally, we present evidence that T lymphoblasts from OS animals are less susceptible to IL 2 antagonistic regulation than normal cells, which possibly further contributes to the T cell hyperfunction of this autoimmune strain.     

Characterization of cellular and molecular immune effectors against Trichinella spiralis newborn larvae in vivo.

The cellular and molecular immune effectors that participated in host immunity against Trichinella spiralis newborn larvae were characterized in vivo using AO rats. Donor rats were immunized with 2,000 muscle larvae orally or 11,400 newborn larvae i.v. Immune serum and cells from spleen, peripheral lymph nodes, mesenteric lymph node, thoracic duct lymph and the peritoneal cavity were obtained from donor rats 10-21 days after infection and transferred into normal recipient rats. The control recipients received either no cells and serum or normal cells and normal serum obtained from normal donors. Newborn larvae (20,000-50,000) were injected either i.v. or ip into these recipients and immunity against newborn larvae was measured either by muscle larvae burden of the recipients three weeks later or by direct recovery of newborn larvae from the peritoneal cavity of the recipients. The experiments demonstrated that immune lymphocytes conferred no protection in the recipients but that immune serum and immune peritoneal cells were protective and these effects were synergistic. Cell adherence to the cuticle and killing of newborn larvae were observed in the peritoneal cavity of immune rats. Positive fluorescence was observed on newborn larvae incubated with fractionated IgM and IgG(E) antibody isotypes. Massive deposition of antibody molecules on newborn larvae was demonstrated by scanning electron microscopy. Studies using transmission electron microscopy revealed that the larval adherent cells were stimulated macrophages, neutrophils and eosinophils.     

Cellular composition of spleen and peritoneal exudate in mice after injection of cyclophosphamide-treated L 1210 leukemia cells

The composition of peritoneal exudate and spleen cells of CD2F1 mice after fourfold i.p. administration of L 1210 leukemia cells treated with cyclophosphamide (L 1210-CY cells) were examined. The number of cells in peritoneal cavity did not increase, however, the spleen weight rose after administration of L 1210-CY cells. The per cent of lymphocytes T was increased 2.5 times but the content of macrophages and lymphocytes B was normal in the peritoneal cavity after L 1210-CY cells injections. In the spleen an 1.4 times increase of the per cent of lymphocytes B, but normal level of macrophages and lymphocytes T were observed.     

Depression of in vitro responsiveness to phytohemagglutinin in spleen cells cultured from chickens with Marek's disease.

Cultures of dispersed spleen cells, prepared from MDV-infected chickens with MD visceral lymphomas, showed marked depression of responsiveness to the T cell mitogen PHA, as measured by 3H-Tdr incorporation in cells in vitro. When data are expressed quantitatively in terms of cpm/10(5) viable cells, the functional depletion of PHA-responsive cells appear to result from lower levels of 3H-Tdr incorporation in the PHA-stimulated spleen cultures from chickens with acute MD symptoms, as compared to similar cultures from uninfected isolator-reared control chickens. It is suggested that depression of PHA-induced blastogenesis is spleen cell cultures from chickens with acute MD reflects virus-related alterations in T lymphocytes.     

Generation of specific helper cells and suppressor cells in vitro for the IgE and IgG antibody responses.

Normal splenic lymphocytes from BDF1 mice were cultured on ovalbumin (OA)-bearing syngeneic peritoneal adherent cells for 5 days and their subsequent helper function was tested by an adoptive transfer technique. Lymphocytes harvested from the culture were mixed with DNP-KLH-primed spleen cells and transferred into irradiated syngeneic mice followed by challenge with DNP-OA. The results showed that the cultured lymphocytes has helper function for both IgE and IgG anti-DNP antibody responses. Depletion of mast cells and T cells in the peritoneal adherent cell preparations did not affect the generation of helper cells in the culture. The helper function of the cultured lymphocytes was abolished by the treatment with anti-theta antiserum and complement and was specific for ovalbumin. The OA-specific helper T cells were generated in vitro by the culture of a T cell-rich fraction of normal spleen on T cell-depleted OA-bearing peritoneal cells. If the normal splenic lymphocytes or T cell-rich fraction were cultured with 10 mug/ml of OA in the absence of macrophages, cultured lymphocytes lacked helper function. The transfer of splenic lymphocytes or splenic T cells cultured with soluble OA to normal non-irradiated mice, however, suppressed both IgG and IgE antibody responses of the recipients to subsequent immunization with DNP-OA. The suppressor cells were sensitive to anti-theta antiserum and complement and their activity was specific for OA. The cultured cells transferred into normal mice did not suppress anti-hapten antibody response to DNP-KLH. Normal lymphocytes cultured on OA-bearing macrophages and had helper function in adoptive transfer experiments failed to suppress antibody response of non-irradiated recipients to DNP-OA. The results indicate that OA-bearing macrophages primed T cells and generated helper T cells, whereas the culture of normal lymphocytes with soluble OA in the absence of macrophages generated suppressor T cells.     

The effect of allogeneic lymphocytes on colony formation during culturing of hematopoietic precursor cells in the peritoneal cavity

The number of colonies formed in the peritoneal cavity (on the artificial underlayer made of peritoneal cells) and in the spleen of lethally irradiated recipients, (CBA X X C57BL) F1 mice, after the intraperitoneal injection of marrow cells depends on the cell donor's genotype: syngeneic cells and cells from mice of the parent strain CBA form fewer colonies in the peritoneal cavity than in the spleen, while cells from C57BL mice produce the reverse distribution of colonies between the peritoneal cavity and the spleen. Allogenic lymphocytes, when transplanted simultaneously with hematopoietic cells, suppress colony formation in the peritoneal cavity from day 2 of cultivation and eliminate the already developed foci of hematopoiesis by day 5.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.     

Analyses of muscular dystrophy and Con A deficiency traits in testcross progeny of chickens

Hereditary muscular dystrophic chickens of the Storrs strain possess two genetic disorders, muscular dystrophy (MD) and a deficient concanavalin A (Con A), a T-cell mitogen, mediated splenic lymphocyte blastogenic response. A possible amelioration of the MD phenotype in MD chickens expressing normal Con A was postulated on the basis of progeny segregating for these two traits in F2 genetic analyses. To test this possibility, testcross progeny were examined for segregation of MD and Con A deficiency traits, and for the degree of muscle destruction and Con A deficiency. The data show both traits to be inherited independently as autosomal recessive traits, and do not support any phenotypic modifications occurring in chickens expressing MD with normal Con A. In the testcross progeny, the Con A deficiency disorder is equally deficient in normal and MD progeny, and the degree of muscle destruction as measured by serum creatine phosphokinase levels is equally great in MD chickens with or without the Con A deficiency trait. The reduced numbers of MD chickens in the testcross progeny can be accounted for by chance and probably reflect losses during in ovo development.     

Demonstration of a tumor-associated surface antigen in Marek's disease.

Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. IV. Role of macrophage and its soluble factor in antigen-induced MIF production of immune T lymphocytes.

Histocompatibility-linked restriction of macrophage-T lymphocyte interaction in antigen-induced MIF production by sensitized lymphocytes was examined, by using combinations of inbred strain 2, strain 13, and JY-1 guinea pigs. The effective interaction of the antigen-bearing macrophages with the immune T lymphocytes was observed when the donor of the antigen-bearing macrophages and that of the immune lymphocytes shared Ia antigens of the major histocompatibility complex. Identities of B antigens and S antigens were not important for this cooperation. It was further demonstrated that the previously reported soluble factor derived from LPS-stimulated peritoneal adherent cells (macrophages) could help antigenic activation of the immune lymphocytes across the strain barrier provided a small number of macrophages (0.01%) from syngeneic strain were present. These results show that the presence of macrophages is absolutely required to present antigen to immune T lymphocytes in a genetically restricted manner and the soluble factor from macrophages appears to give a nonspecific effect on the lymphocyte activation in addition to or in collaboration with antigenic stimulation.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.     

双歧杆菌和乳杆菌在诱发抗肿瘤免疫中的作用

双歧杆菌和乳杆菌给封闭群昆明小鼠腹腔注射,在体内激活后,胸腺细胞和脾细胞对ＣｏｎＡ刺激的增殖反应,脾贴附性细胞对ＹＡＣ－１,Ｌ９２９的细胞毒作用,以及脾贴附性细胞产生对上述二株瘤细胞的肿瘤坏死因子（ＴＮＦ）的活性都比对照动物明显增强。结果提示短双歧杆菌和嗜酸性乳杆菌给小鼠腹腔注射后,通过激活脾脏淋巴细胞和贴附性细胞（巨噬细胞）所介导的免疫功能而明显地增强宿主的抗肿瘤活性。     

Genetic variation in the recruitment and activation of chicken peritoneal macrophages

Genetic variation in the ability to recruit and activate peritoneal macrophages was examined in seven partially developed 15I5-B congenic White Leghorn chicken lines. While the ability to generate peritoneal exudate cells (PECs) was similar in all lines, major differences were observed in the numbers, composition, and functional activity of harvestable peritoneal adherent cell populations. In response to a general stimulant, Sephadex, lines .7-2 and .6-2 produced the greatest numbers of adherent peritoneal cells while lines .C-12 and .15I-5 were among the poorest responders. Macrophage percentage of adherent PECs varied between lines. 15I5 chickens produced a consistently high percentage of adherent macrophages while .6-2 birds exhibited the lowest macrophage percentage at all ages examined. Phagocytosis was used as one measure of the level of macrophage activation and similar results were obtained using both opsonized and unopsonized sheep erythrocytes; adherent peritoneal cells from lines .6-2, .7-2, and .P-13 exhibited the highest activity and .C-12, .15I-5, and background 15I5(B15) lines produced cells with the lowest phagocytic activity. In a second functional assay, the killing of Salmonella typhimurium, macrophage-rich cells from line .P-13 exhibited the lowest activity which was significantly lower than that obtained with cells from lines .6-2 and .15I-5. Antigen-specific stimulation of peritoneal adherent cells by ferritin also showed that .C-12 was a low responder in contrast with other lines. The results indicate that these genetic lines differ in peritoneal macrophage function and suggest that the chicken major histocompatibility complex may influence certain properties of chicken macrophage function.