Production and characterization of monoclonal antibodies against chicken lymphocyte surface antigens

A panel of monoclonal antibodies (mAbs) with specificity for chicken lymphocyte surface antigens was established and characterized based on their reactivities against chicken lymphoid cells and tumor cell lines on flow cytometry. Three mAbs (7-3G-2, 7-2E-8, and JB-2) reacted preferentially with thymocytes, however, none of them reacted with Marek's disease derived T lymphoblastoid cell lines. Four mAbs (6-27A-1, 4-5C-5, Lc-4, and Lc-6) reacted with spleen cells and peripheral blood leukocytes as well as thymocytes. All seven mAbs reacted with chicken embryonic thymocytes from day 12 of embryonic life onward. All mAbs showed no reactivity against bursal lymphocytes.     

Immune system of swine: dissection of mononuclear leucocyte subpopulations by means of two-colour cytofluorometric analysis

Expression of the differentiation antigen 8/1, a porcine leucocyte marker for which no counterpart is known as yet in other species, was tested in relation to the expression of surface Ig and the monocyte marker 74-22-15. B lymphocytes and T lymphocytes can be typified by the exclusive expression of surface Ig and 8/1, respectively, while all monocytes defined by the expression of the 74-22-15 antigen co-express 8/1 with the highest staining density observed for this marker. 8/1+ T lymphocyte subsets defined by the expression of CD4 and CD8 cell surface antigens differed in the expression of 8/1 with the staining density being high in CD4+ T lymphocytes, intermediate in CD8+ as well as in CD4+CD8+ T lymphocytes, and low in the CD4-CD8- subset. Practically all mononuclear leucocytes present in peripheral blood are included in the subpopulations defined by this kit of reagents, and thus it can be predicted that if mononuclear leucocytes which do not belong to any of these subpopulations exist at all, these must be rare and will not be accessible to an analysis without enrichment.     

Identification of bovine lymphocyte subpopulations by a combined bacterial adherence and fluorescent antibody technique

It is known that certain strains of bacteria bind selectively to subpopulations of human peripheral blood lymphocytes. We have developed a technique which used the specificity of bacterial binding concurrently with fluorescent antibody staining methods to identify 5 B-cell and 5 T-cell subpopulations of bovine lymphocytes. In addition, greater than 95% of the peripheral blood lymphocytes could be positively identified as being either T-cells or B cells. Using ethidium bromide-stained bacteria and lymphocytes in combination with fluorescent antibody staining to detect surface immunoglobulins or T-cell antigens, the method provided a simple yet highly specific technique for the enumeration of both B and T cells in 1 preparation of peripheral blood lymphocytes. The use of bacterial rosetting with fluorescent antibody staining was found to be easier and more reliable than the methods currently used to identify bovine B- and T-lymphocyte subpopulations.     

Influence of chronic caprine arthritis-encephalitis virus infection on the population of peripheral blood leukocytes

The influence of caprine arthritis-encephalitis (CAE) virus infection on the population of peripheral blood leukocytes in goats was evaluated. For this purpose two groups of adult dairy female goats were formed. The experimental group consisted of 17 goats, which had been naturally infected for many years. The control group comprised 29 non-infected goats, which originated from CAE-free herd. All goats were clinically healthy. Whole blood was collected and tested in hematological analyzer and light microscope to assess the total number of leukocytes and the percentage of four leukocyte populations--neutrophils, eosinophils, monocytes and lymphocytes. Then, flow cytometry with monoclonal antibodies against several surface antigens (namely CD14, CD2, B-B2, CD4, CD8h, TCR-N6, WC1-N2 and WC1-N3) was performed to assess the proportion of lymphocyte subpopulations. Statistically significant differences (alpha < or = 0.01) were observed only in the subpopulations of T lymphocytes--percentage of all subpopulations were significantly higher in the group of seropositive goats. No statistically significant differences were revealed with respect to the total number of blood leukocytes, the average percentage of blood leukocyte populations and proportions of both T and B lymphocytes.     

Effects of corticosteroids on lymphocyte subpopulations and lymphokine secretion in chickens.

Various effects of glucocorticosteroids on the avian immune system were examined in chickens treated intramuscularly with 0.1 to 2.5 mg dexamethasone or prednisolone. Kinetic changes in body weight gain, percentages of lymphocyte subpopulations, and T-cell functions were examined following treatment with dexamethasone or prednisolone every other day. Chickens treated with dexamethasone or prednisolone showed a decrease in body-weight gain compared with age-matched, untreated chickens. In general, the total number of splenic lymphocytes of chickens treated with the two drugs was significantly lower than in controls in a dose-dependent manner. Flow cytometric analysis of splenic lymphocyte subpopulations revealed that the percentages of lymphocytes expressing CD8, gamma delta T-cell receptor, Ia, or IgM antigens and natural killer cells were lower in dexamethasone-treated chickens than in the controls, whereas the percentages of T lymphocytes bearing CD3, CD4, or alpha beta TCR antigens were higher. Furthermore, splenic T cells obtained from dexamethasone-treated chickens showed a significant depression in concanavalin A-induced lymphoproliferation and interleukin 2 and gamma-interferon production. The results characterize a variety of immunosuppressive effects of glucocorticoids on the avian immune system.     

The bovine lymphoid system: II. Derivation and partial characterlzation of monoclonal antibodies against bovine peripheral blood lymphocytes

We have raised monoclonal antibodies to produce reagents specific for bovine lymphocyte subpopulations. Spleen cells from mice immunized with bovine peripheral blood lymphocytes were fused with X63-Ag8 myeloma cells and eleven myeloma-hybrids which secreted antibody specific for bovine lymphocytes were doubly cloned. Five of the hybrids secreted antibodies which bound to the majority of bovine lymphocytes. Two of these antibodies were specific for polymorphic antigens. One antibody bound to B lymphocytes and serum IgM molecules. The remaining five bound to subpopulations of lymphocytes. Four monoclonal antibodies bound only to bovine cells while six also bound to lymphocytes from other bovidae. None bound to human lymphocytes. We discuss the difficulty of correlating the specificities of monoclonal antibodies to functional lymphocyte subpopulations in outbred animals where few other defined markers are available.     

Effect of dietary n-6-to-n-3 fatty acid ratio on complete blood and total white blood cell counts, and T-cell subpopulations in aged dogs

OBJECTIVE: To determine effect of diets with variable n-6-to-n-3 fatty acid (FA) ratio on CD4+ and CD8+ T-lymphocyte subpopulations, and on results of routine laboratory analyses (CBC and total WBC count, serum biochemical analyses, and urinalysis). ANIMALS: 20 healthy, aged (9.5 to 11.5 years old) female Beagles. PROCEDURE: Dogs were fed 1 of 3 diets that contained 6% fat by weight but differed in amounts of n-6 and n-3 FA. For 11 weeks, 6 dogs were fed a low concentration of n-3 FA (ratio, 31:1), 7 were fed a medium concentration (5.4:1), and 7 were fed a high concentration (1.4:1). Preprandial blood and urine samples were collected before beginning the study and at 8 weeks for evaluation of laboratory variables. Before and at 3, 6, and 8 weeks during the study, blood was drawn for total WBC and lymphocyte counts and for characterization of T-cell subpopulations. At 8 and 10 weeks, dogs were vaccinated with keyhole limpet hemocyanin suspension. Blood was drawn 4 days after each vaccination, and lymphocytes were isolated for flow cytometry. Effects of diet and vaccination on each variable were determined. RESULTS: After vaccination, total lymphocyte count increased and CD4+ T lymphocyte count and the CD4(+)-to-CD8+ ratio decreased in dogs consuming the diet with n-6-to-n-3 FA ratio of 1.4:1. CONCLUSION: Feeding a diet with n-6-to-n-3 FA ratio of 1.4:1 had significant effects on CD4+ T lymphocytes in healthy, aged Beagles after vaccination.     

Immunophenotypical characterization of lymphocyte subpopulations of the uterus of non-pregnant and pregnant goats

The increased susceptibility during pregnancy to certain pathogens that cause abortions may be related to changes in the distribution and phenotype of lymphocyte subpopulations in the uterus. Histological, electron microscopic and immunocytochemical techniques were used in this study to examine whether such variations occur in different stages of the reproductive cycle of goats. The study of non-pregnant goats showed that most uterine lymphocytes were T cells and displayed both an intraepithelial and stromal distribution. CD8+ T lymphocytes were more numerous than CD4+ T lymphocytes. In the endometrial epithelium two lymphocyte subpopulations were observed: non-granulated CD2+ CD8+ T lymphocytes and granulated CD2+ CD8- T lymphocytes. During gestation, no lymphocytes were observed in the placentomal area, while a decreased number of T lymphocyte subpopulations were found in the inter-placentomal area. In the inter-caruncular epithelium, non-granulated CD2+ CD8+ T lymphocytes disappeared, whereas the granulated CD2+ CD8- T lymphocyte subpopulations increased their number and changed their morphology.     

Changes in peripheral blood leucocyte counts and subpopulations after experimental infection with BVDV and/or Mannheimia haemolytica

Leucocyte counts and subpopulations were studied in peripheral blood from calves experimentally infected in the respiratory tract with either bovine virus diarrhoea virus (BVDV) or Mannheimia haemolytica (Mh), or with a combination of both agents (BVDV/Mh). A non-inoculated control group was included. Peripheral blood samples were obtained for total leucocyte counts, and for neutrophil, lymphocyte and monocyte counts. The numbers of blood lymphocytes expressing the surface antigens CD4, CD8, WC1, B and IL-2R were analysed using flow cytometry. The results showed that BVDV inoculation induced a significant decrease in total leucocyte counts and in neutrophil and lymphocyte numbers, while Mh inoculation induced significant increases in total leucocyte counts and neutrophils, while the lymphocyte count decreased. In the BVDV/Mh group, the total leucocyte count and the lymphocyte numbers decreased significantly. In this group, the lymphocyte numbers remained on a very low level throughout the rest of the study. The numbers of CD4+, CD8+ and WC1+ lymphocytes decreased significantly compared with before inoculations mainly in the BVDV and BVDV/Mh groups. The drops were most pronounced in the BVDV/Mh group. The numbers of B+ lymphocytes and IL-2R+ cells did not change significantly.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.     

Characterization of monoclonal antibodies to feline T lymphocytes and their use in the analysis of lymphocyte tissue distribution in the cat.

We describe the development of three monoclonal antibodies to feline T lymphocytes. Antibody 1.572 stains 93% of feline thymocytes, 49% of lymph node, and 65% of spleen lymphocytes. Two-color analysis shows 1.572 does not stain Ig-bearing cells, and 1.572-positive lymphocytes plus Ig-positive lymphocytes make up approximately 90% of peripheral blood lymphocytes (PBL), suggesting that 1.572 is a pan-T cell marker. The other two monoclonal antibodies, 3.357 and CAT30A, stain a smaller population of thymocytes (59%) of which 40% express both antigens. The 3.357 antigen is found on 23% of lymph node and 47% of spleen lymphocytes, while the CAT30A antigen is found on 29% of lymph node and 19% of spleen lymphocytes. Two-color analysis shows that 3.357 and CAT30A stain mutually exclusive subpopulations of 1.572-positive cells. Using thymocytes as an antigen source, antibody 3.357 precipitated a molecule of 66,000 molecular weight (Mw) under nonreducing conditions and a heterodimer of 32,000 and 34,000 under reducing conditions, suggesting that 3.357 recognizes the feline CD8 homologue. Antibody CAT30A precipitated a molecule of 55,000 Mw under both reducing and nonreducing conditions, which suggests it recognizes the feline CD4 homologue. Analysis of PBL profiles of 35 normal cats using the three monoclonal antibodies indicates that the distribution of feline PBL subpopulations is similar to man, including the CAT30A:3.357 ratio (1.74), which is identical to reported CD4:CD8 ratios in man. Based on these data, the feline CD4 and CD8 homologues are similar to those reported in other species.     

猪瘟病毒感染对猪外周血T淋巴细胞亚群及TNF-α和IFN-γ的影响

为探讨猪瘟病毒(CSFV)弱毒株T株和野毒株G株感染对猪外周血T淋巴细胞亚群、TNF-α和IFN-γ的影响,本研究应用流式细胞术和ELISA等方法检测CSFV感染猪与未感染猪的白细胞凋亡、CD4+与CD8+T淋巴细胞亚群数量的动态变化以及TNF-α和IFN-γ的动态变化。结果表明,猪感染CSFV T株和G株第4 d和第7 d后CD4+T淋巴细胞比例分别为28.6%、26%和26%、20%,未感染前分别为33.4%和36.8%。猪感染CSFV T株和G株第4 d、第7 d后CD8+T淋巴细胞比例分别为41%、32%和38%、25%,感染前分别为43.8%和48.8%。外周血白细胞凋亡的检测结果显示,猪感染CSFV T株和G株第7 d后,白细胞凋亡比例分别为8.35%和9.89%,未感染的猪为1.63%。ELISA检测结果表明,猪感染CSFV T株和G株第7 d后,TNF-α的产生量分别为553.4 pg/mL和594.2 pg/mL;IFN-γ的产生量分别为8.2 pg/mL和9.8 pg/mL,未感染猪分别为498 pg/mL、12.5 pg/mL。以上结果提示,CSFV感染会引起机体免疫相关细胞及免疫分子发...     

In vitro stimulation of pig lymphocytes after infection and vaccination with Aujeszky's disease virus

Mitogenic and antigenic lymphocyte stimulation were examined in Aujeszky's disease virus (ADV) infected pigs and in pigs vaccinated with modified live ADV. Neither infection nor vaccination had any effect on lymphocyte responsiveness to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) or concavalin A (Con A). ADV antigen-responsive lymphocytes began to appear in the peripheral blood between 7 and 14 days after inoculation and could still be demonstrated in blood and spleen of infected pigs at 174 days after infection. In vaccinated pigs, sensitized peripheral blood lymphocytes could be detected up to at least 35 days after revaccination. Pre-incubation of ADV antigen with specific antibody markedly reduced lymphocyte stimulation. Non-immunized pigs showed no lymphocyte response to ADV antigen. Infected pigs exhibited no lymphocyte reactivity against antigens of non-infected cells.     

Enumeration of lymphocyte subsets in peripheral blood of rhesus monkeys prior to and following monocyte removal using a modified indirect avidin-biotin immunoperoxidase technique

A modified avidin-biotin immunoperoxidase technique has been used to enumerate B lymphocytes, T inducer/helper (TH) and T cytotoxic/suppressor (TS) cells in the peripheral blood of normal and immunosuppressed rhesus monkeys (Macaca mulatta) prior to and following adherent-cell depletion. The levels of each of the B, TH and TS cells detected in the normal monkeys using monoclonal antibodies which recognized specific surface antigens on human lymphocytes were comparable to the levels reported in human peripheral blood using direct immunofluorescence, immunoperoxidase or flow cytometry techniques. Adherent cell depletion did not result in a significant loss of any of the lymphocyte subpopulations examined. The technique is reproducible and sensitive in detecting differences between normal and immunosuppressed animals, and would prove to be useful in studies pertaining to chemical and drug immunomodulation.     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

Dynamics of lymphocyte subpopulation changes in the cecal tonsils of chickens infected with Salmonella enteritidis

Salmonella enteritidis (SE)-induced changes in various T and B lymphocyte subpopulations in the cecal tonsils of chickens were analyzed using flow cytometry. At 1 day post-SE inoculation, the percentages of CD3(+) and CD8(+) T lymphocytes were significantly decreased in the group inoculated with 1x10(9) SE colony-forming units (CFU) (SE high) and in the group inoculated with 1x10(6) SE CFU (SE low) compared with the uninfected control group. The percentage of CD4(+) T lymphocytes was significantly increased in the SE high group compared to the uninfected and the SE low groups at 4 days after SE inoculation. The percentage of IgG(+) B lymphocytes was also significantly increased in both SE high and low groups compared to the uninfected control at 6 days post-SE inoculation. In contrast, the SE low group showed significantly fewer IgM(+) B lymphocytes compared to the uninfected and SE high groups. These results show that SE infection induces significant changes in the cecal tonsil lymphocytes subpopulations shortly following SE inoculation.     

E.tenella初次感染雏鸡外周血T淋巴细胞亚群及增殖能力变化的动态研究

应用流式细胞术（ FCM）和MTS比色法检测雏鸡初次感染柔嫩艾美尔球虫（ E． tenella）后外周血中CD3＋CD4＋、CD3＋CD8＋T淋巴细胞及其增殖能力的动态变化。数据显示,E． tenella初次感染雏鸡后7～20 d CD3＋CD4＋、CD3＋CD8＋T 淋巴细胞比例明显高于对照组雏鸡（ P＜0．05和P＜0．01）,并且均于12 d时达到峰值。初次感染后7～20 d雏鸡外周血中CD4＋／CD8＋T淋巴细胞亚群的比值明显升高,于感染后16 d时达到1．78。 E． tenella初次感染雏鸡后12～20 d外周血淋巴细胞增殖能力明显高于对照组雏鸡（ P＜0．05）,并于16 d达到峰值。试验表明E． tenella初次感染雏鸡能激活淋巴细胞产生增殖反应,CD3＋CD4＋、CD3＋CD8＋T淋巴细胞在抵抗E． tenella初次感染过程中有重要作用。     

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.     

Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: individual variability and repeatability

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.