Rosetting of bovine T-lymphocytes with autologous and allogeneic red blood cells

Certain bovine peripheral blood lymphocytes (PBL) and foetal thymocytes were shown to bind autologous and allogeneic red blood cells (RBC). When autologous RBC were treated with dextran, approximately 10% of peripheral blood lymphocytes and about 30% of thymocytes were found to form rosettes. Cells forming autologous rosettes appear to be a population of T-lymphocytes because (1) more rosette formation occurred with thymocytes than with PBL, (2) autologous rosette formation was increased in PBL cultures enriched in T cells and was decreased in cultures depleted of T cells, (3) very few rosette forming cells had surface immunoglobulin and (4) peripheral blood mononuclear cell cultures depleted of monocytes did not show a decreased autologous rosette formation. It appears that the cells forming rosettes with autologous and allogeneic RBC belong to the same sub-population of T-cells.     

Staining of pig lymphocytes subpopulations with acid alpha-naphthyl acetate esterase

The use of alpha-naphthyl acetate esterase (ANAE) as a T cell marker in some other species and the broad correlation of incidence of ANAE-positivity and E rosette-formation in the pig suggest that ANAE-staining may be a T-cell marker in the pig. However, by studying the staining of lymphocytes within a variety of rosettes in fixed preparations a similar incidence of pig blood lymphocytes were found to be ANAE+ among T cells (E rosettes formed in dextran), B cells (antiglobulin rosettes) and Fc-gamma receptor-bearing B and T cells (EA rosettes in saline and dextran): complement (C') receptor-bearing cells showed a higher incidence of staining than other lymphocytes. Analysis of staining morphology suggested that certain morphologies within the B and T lineages may be confined to subpopulations. Thus ANAE positivity is certainly not a marker identifying blood T lymphocytes but could be of some value indicating subpopulations of B and T lymphocytes.     

Surface markers for characterisation of bovine blood lymphocyte populations and changes in these from birth to maturity

A sheep erythrocyte (E) rosette technique was developed for use with cattle lymphocytes. This involved the use of 17 per cent Ficoll 400 and preservative-free heparin (84 iu/ml) in the saline-erythrocyte mixture. Using this technique, 83 per cent of peripheral blood lymphocytes in cattle aged between six and 10 years were found to form E rosettes. The remaining cells (17 per cent) were B-cells, so that no cells remained unmarked. Lymphocytes from very young calves contained a population of unmarked or null cells, but these rapidly diminished as the animals matured. A peak of total lymphocytes recovered from blood, as well as E rosette-forming cells, occurred in calves aged four to six months. The non-E rosette-forming cells were mostly B-cells and it was suggested that this was associated with calf weaning. The total number of lymphocytes recovered, as well as E rosette-forming cells, gradually fell with the age of the cattle sampled. Null cells were virtually absent from the blood of cattle six years and older. Bovine T-cells could be further subdivided into Fc mu, Fc gamma and C' receptor-bearing subpopulations on the basis of overlap with R rosette-forming cells. Some further separation of these cells from B-cells was achieved using density gradient centrifugation on Percoll. Separation of E rosette-forming cells with Fc receptors from E rosette-forming cells without Fc receptors was achieved by nylon wool columns, to which the Fc receptor bearing cells were adherent. It was concluded that bovine blood lymphocytes had blood T-lymphocyte populations with markers which may correspond to the 'helper' (Fc mu ) and 'suppressor' (Fc gamma ) populations described for the human.     

Characterization of lymphocyte subpopulations in sheep by rosette formation,adherence to nylon wool and mitogen responsiveness

Sheep peripheral blood lymphocytes have been studied using a number of surface markers. Thus 16.6 ± 2.4% (mean ± S.E.) were surface immunoglobulin positive (sIg⁺) by direct immunofluorescence, 35.9 ± 2.1% formed Fc rosettes with bovine red blood cells (RBC) sensitized with rabbit antibody (Fc⁺) and 28.4 ± 2.0% formed rosettes with sheep red blood cells (RBC) in the presence of 4% dextran (DS⁺). The percentage of both Fc⁺ and DS⁺ lymphocytes tended to increase with age of the animals. Demonstration of these markers allowed computation of two further subpopulations: null cells lacking sIg and a receptor for sheep RBC, and Fc·null cells lacking a receptor for Fc and sheep RBC. The former population, which contained a proportion of Fc⁺ lymphocytes comprised 49.8 ± 3.8% of blood lymphocytes and the latter 38.4 ± 3.0%.Separation on nylon wool columns, selective rosette enrichment and depletion on density gradients and stimulation with phytomitogens have shown sIg⁺ and Fc⁺ lymphocytes to be nylon wool adherent and unresponsive to phytohaemagglutinin (PHA) and Concanavalin A (Con A) and DS⁺ lymphocytes to be nylon wool non-adherent and responsive to PHA and Con A. The data also indicates a major overlap of the lymphocyte subpopulations bearing sIg and Fc which are apparently B lymphocytes. Moreover these data support the contention that E-rosette formation with sheep RBC in the presence of dextran is a marker for sheep T cells. The data also indicates that Fc·null cells are T cells, eluting in the non-adherent fraction from nylon wool. It is probable that a proportion of these cells bear a SRBC receptor too weak for present detection methods.     

Characterization of feline T and B cells

Feline peripheral-blood lymphocyte populations (n = 22) were examined for the following markers: rosette formation with guinea pig erythrocytes (GPE-T cells), rosette formation with human RBC (HRBC-T cells), rosette formation with sheep RBC, mixed rosette formation with GPE-T cells and HRBC-T cells (total T cells), erythrocyte antibody-complement rosettes, and surface immunoglobulin. An average of 28% +/- 7% (range, 16% to 39%) of the feline lymphocytes formed rosettes with GPE-T cells, and 27% +/- 7% (range, 11% to 36%), with HRBC-T cells. An average of 57% +/- 9% (range, 33% to 75%) of the lymphocytes formed mixed rosettes. The erythrocyte antibody-complement rosette-forming cells and surface immunoglobulin-bearing cells were found in peripheral blood lymphocytes (10% +/- 6% and 24% +/- 8%, respectively). The murine monoclonal antibodies OKT 11 and HuLy-m1, specific for a framework determinant of human E-rosette receptor antigens, cross-reacted with feline cell membrane molecules recognizing a bimolecular complex (45,000 to 50,000 daltons) similar to that described in persons. We investigated the distribution of these E-rosette receptor-like antigens on feline lymphocytes. By complement-mediated lymphocytotoxicity, about 30% of the feline lymphocytes expressed the antigens. When lymphocytes were treated with HuLy-m1 antibody, spontaneous rosette formation with HRBC-T cells was significantly inhibited.     

Erythrocyte rosettes--a marker for bovine T cells.

Many species of erythrocytes were investigated for their ability to form spontaneous rosette with bovine peripheral blood leukocytes and fetal thymocytes. Only sheep and chicken red blood cells gave rosettes. Using conditions shown optimum for the demonstration of human rosette forming cells, only low numbers of bovine rosettes were demonstrable. By changing culture conditions to include 100% fetal calf serum, neuraminidase treated erythrocytes and/or lymphocytes and optimizing the incubation times and temperature, up to 38% of peripheral blood leukocytes and 52% of thymocytes formed rosettes. A thymic origin of rosetting cells was ascribed to T cells for the following reasons: 1) thymocytes gave higher numbers than did peripheral blood leukocytes, 2) rosette forming cell numbers were increased in peripheral blood leukocyte subpopulations enriched in T cells by nylon column separation and 3) only very few rosette forming cells had surface immunoglobulin, a marker of B lymphocytes. The reasons why all T cells were not detected by the technique were discussed.     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

Immunologic surface markers on nonhuman primate lymphocytes.

Peripheral blood lymphocytes from 37 healthy rhesus macaques (Macaca mulatta) and thymocytes from 10 fetal and neonatal rhesus macaques were studied for membrane characteristics. Spontaneous rosette formation with sheep erythrocytes, a characteristic of human T lymphocytes, was evaluated. The presence of membrane-bound immunoglobulin and surface receptors for fixed complement was measured, using fluorescent antibody techniques and erythrocyte-antibody-complement rosettes, respectively. The mean percentages +/- 1 standard error of the lymphocyte markers in the peripheral blood lymphocytes from the macaques were: spontaneous rosettes, 63 +/- 1.0; erythrocyte-antibody-complement rosettes, 14.9 +/- 1.2; and membrane immunoglobulin-positive cells, 21.9 +/- 2.2. These values are very similar to values reported for human beings.     

Distribution of T and B cells in thymus, blood and lymph nodes of the cat.

The proportions of T and B cells in the thymus, blood and lymph nodes were estimated in cats from three months to three years of age. T cells were identified by formation of E rosettes with guinea-pig red blood cells and B cells by the presence of surface immunoglobulin (Ig) as shown by the mixed antiglobulin reaction. Using a double test, it was shown that these methods identify separate, non-overlapping cell populations. The proportions of T cells shown in the thymus ranged from 10-73 per cent, in the blood 5-62 per cent and in the nodes, 5-44 per cent. Cells with surface Ig ranged from 0-10 per cent in the thymus, 26-68 per cent in the blood and 32-18 per cent in the nodes. Two cats with lymphadenopathy had unusually high B cell counts and one cat with depletion of the thymus was deficient in peripheral T cells. Papain treatment reduced or abolished E rosette formation by T cells.     

Role of the spleen and rosette-formation response in experimental Eperythrozoon ovis infection

The role of the spleen and rosette-formation responses was investigated in sheep experimentally infected with Eperythrozoon ovis. Phagocytic activity was observed in the spleen 19 days after primary infection. Phagocytosis of E. ovis-parasitised and non-parasitised erythrocytes by cordal reticular cells occurred. E. ovis organisms seemed to be detached from the erythrocytes by pseudopodia extending from macrophages and cordal reticular cells without causing damage to the plasmalemma of the erythrocyte. No phagocytic activity was observed in spleens removed 74 and 146 days after infection. Antigen-specific lymphoid cell responsiveness, assessed by rosette formation, indicated that 2.8, 15.4, 8.0 and 6.0% of lymphoid cells in the spleens of the four E. ovis-infected sheep, respectively, formed antigen-specific rosettes. Rosette formation did not occur when splenic lymphocytes from E. ovis-infected sheep were mixed with non-infected erythrocytes or when splenic lymphocytes from an uninfected sheep were used.     

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fc positive cells was assayed by the EA rosette test.The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4±1.6%), as detected by E rosettes, was significantly (p<0.01) higher than in control animals (42.4±3.5%). In clinically healthy horses 8.9±1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p<0.001) percentage of these cells (4.7±0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p<0.001) in EIA-infected horses (24.7±0.8% and 42.8±2.2% respectively) as compared to uninfected animals (15.1±1.4% and 29.6±1.2% respectively). Receptors for mouse erythrocytes, as yet undescribed on equine PBL, were demonstrated in approximately equal proportions on lymphocytes from EIA-infected (24.8±1.5%) and uninfected horses (24.3±2.1%).     

Rosette formation by buffalo (Bos bubalis) T and B lymphocytes.

Buffalo (Bos bubalis) lymphocytes were purified and tested for their E and EAC rosette forming capacity as a marker for the detection of T and B cells, respectively. Sheep erythrocytes were found to form 17.7 per cent of E rosette with buffalo lymphocytes. This population of lymphocytes is believed to be T cell. Erythrocytes of guinea-pig, rabbit, hamster, rat, chicken, dog and donkey formed a lower percentage of rosettes. Five to 18.5 per cent of SRBC-EAC rosettes were detected with buffalo lymphocytes which are believed to be B cells.     

Cell surface markers on canine lymphocytes.

Reference values for T and B lymphocytes were determined on lymphocytes from canine thymus, spleen, lymph node, bone marrow, and peripheral blood by use of erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette assays, plus a direct fluorescent technique for assay of surface immunoglobulins. Numbers of T lymphocytes, indicated by E rosette formation with human erythrocytes, ranged from a low of 1% in the thymus to 13% in the peripheral blood, whereas B-lymphocyte numbers ranged from 3% (thymus) to 41% (bone marrow) and from 6% (thymus) to 36% (bone marrow), as indicated by EAC rosette formation or presence of surface immunoglobulins respectively. Stimulation of peripheral blood lymphocytes with either phytohemagglutinin or concanavalin A increased the total number of E-rosetting cells two to threefold, whereas the number of EAC-rosetting cells decreased by half. Further, the percentage of cells bearing Fc receptors increased after phytohemagglutinin stimulation. These results indicate the E rosette technique can be used to identify and to monitor a population of canine T lymphocytes.     

猪囊虫病基因工程疫苗的体液与细胞免疫反应

为了探讨以 Ｉ Ｓ Ｃ Ｏ Ｍ 作佐剂的猪囊虫病基因工程疫苗的免疫机理,对其诱导的体液免疫与细胞免疫反应进行了测定。用上述疫苗免疫 ９ 头试验猪,采用间接 Ｅ Ｌ Ｉ Ｓ Ａ 检测体液免疫反应及通过淋巴细胞转化试验、 Ａ Ｎ Ａ Ｅ染色试验、 Ｅ玫瑰花环形成试验等检测细胞免疫反应;用该疫苗和铝胶苗分别免疫昆明小鼠各 ２０ 只,分别检测体液免疫反应和 Ｔ 淋巴细胞抑制／杀伤亚群的动态变化。体液免疫的检测结果显示,免疫后 ７ 天即出现抗体,２１ 天后抗体全部转阳,持续的时间不少于 １９３ 天,效价明显高于铝胶苗;细胞免疫检测结果显示,免疫猪外周血 Ｔ淋巴细胞转化率、 Ａ Ｎ Ａ Ｅ＋ 细胞和粗粒型 Ａ Ｎ Ａ Ｅ＋ 细胞、 Ｅ Ｒ Ｆ Ｃ和 Ｅａ Ｒ Ｆ Ｃ细胞显著升高,免疫小鼠 Ｔ淋巴细胞抑制／杀伤亚群显著升高;与铝胶苗及对照组比较,差异极显著。以上结果表明猪囊虫病基因工程疫苗可同时激发动物的体液免疫和细胞免疫反应,增强了机体的免疫调节功能及杀伤性 Ｔ 淋巴细胞功能。     

Characterization and separation of bovine lymphocyte populations

Bovine lymphocyte populations were characterized by surface markers, rosette-forming ability and behaviour towards mitogens. After pre-treatment with neuraminidase 16% of the bovine blood lymphocytes and 14% of the bovine spleen cells formed spontaneous (E) rosettes with sheep erythrocytes. About 20% EAC rosette-forming cells were detected among both cell populations. Protein A receptors were detectable among 8% of the blood lymphocytes and 26% of the spleen cells. Bovine lymphocytes responded to pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (Con A). An enrichment of bovine B and T cells was obtained by E-rosette sedimentation (81–84% B cells) and by filtration through nylon fiber columns (51–65% T cells). The T cells obtained after nylon filtration still responded to the mitogens PHA, Con A and PWM. Enriched B-cell populations responded to bacterial lipopolysaccharide (LPS). After monocyte depletion the mitogenic response of blood lymphocytes was not influenced.     

A monoclonal antibody specific for bovine T cell surface antigen associated with the E-rosette receptor

From mice immunized with T lymphocyte-enriched bovine peripheral blood mononuclear cells (PBMC), a monoclonal antibody termed BLMo-12 was obtained. BLMo-12 reacted with the antigen of Mr 56,000 in lysate of T lymphocytes. This mAb was found to inhibit spontaneous rosette formation by T-bovine lymphocytes with sheep red blood cells but it did not react with B lymphocytes, monocytes, neutrophils or eosinophils. In frozen section of the thymus, BLMo-12 showed a positive staining both the cortex and the medulla. In lymph nodes, the mAb stained the T-dependent paracortex. BLMo-12 reacted with 49.9% of PBMC and 82.5% of thymocytes. Recognition of the bovine homologue of CD2 on the T lymphocyte surface by this mAb was discussed.     

A study of porcine lymphocyte populations. II. Characterization of porcine lymphocyte populations

The percentage of E rosette forming cells amounted to 26% of the blood lymphocytes and 34% of the spleen cells in German Landrace pigs. 10% of the live lymphocytes in the peripheral blood and 22% of the spleen cells were EAC rosette forming cells. The number of E rosettes could be increased by treatment of sheep erythrocytes with neuraminidase. The number of lymphoid cells reacting with protein A in the peripheral blood and in the spleen of pigs correlated well with the number of EAC rosette forming cells. The mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are potent stimulators of pig lymphoid cells. The mitogenic stimulation of pig lymphocytes could not be influenced significantly by the removal of phagocytic cells. By neuraminidase treatment the mitogen induced stimulation rate was decreased. For the mitogenic stimulation of porcine lymphoid cells in the presence of PHA, Con A and PWM T cells were required. Bacterial lipopolysaccharides (LPS) stimulated only B cells to a small degree.     

Subpopulations of bovine lymphocytes separated by rosetting techniques

A combination of E-rosetting techniques with both neuraminidase- and AET-treated sheep erythrocytes (RBC) was used to enumerate subsets of bovine T lymphocytes. Direct (anti-Ig) rosetting procedures were used to enumerate B lymphocytes bearing surface immunoglobulin (Ig). Approximately 10% of bovine peripheral blood lymphocytes formed rosettes only with neuraminidase-treated RBC; 20% formed rosettes with either neuraminidase- or AET-treated RBC; 30% formed rosettes only with AET-treated RBC; 25% possessed surface Ig, as shown by rosette formation with anti-Ig-coupled RBC; and the remaining 15% lacked both E receptors and surface Ig. These five populations were physically separated by centrifugation on Ficoll-Diatrizoate and recovered for functional analysis. The procedures reported here should be useful for the identification of lymphocyte populations responsible for recognition, effector, and cooperative functions in the bovine immune system.     

Bovine lymphocytes: erythrocyte rosettes in normal, lymphomatous and corticosteroid-treated cattle.

Spontaneous erythrocyte rosettes, antibody-complement rosettes and nonrosetting cells were enumerated for peripheral blood lymphocytes of normal adult, lymphomatous adult and immature cattle as well as for peripheral blood lymphocytes of adult cows both before and after injection of corticosteroids. Calf thymic lymphocytes were also examined for rosette formation. Results indicate significant reduction in peripheral blood lymphocyte-erythrocyte rosettes and nonrosetting cells in tumour-bearing cows with a simultaneous elevation in percent antibody-complement rosettes. Calf thymus had a significantly greater percent erythrocyte rosettes than did peripheral blood lymphocytes from the same individuals. Corticosteroid injection reduced peripheral blood lymphocytes without altering proportion of cells as erythrocyte rosettes, antibody-complement rosettes or nonrosetting cells.     

Horse anti‐bovine lymphocyte serum. Its effect in calves

Summary

Anti‐bovine lymphocyte serum (ABLS) had been prepared in horses with calf thymocytes as antigen and its effects in calves following parenteral administration were studied. The optimal dose was found to be one ml/kg body weight. The A BLS suppressed both the T and B cell functions. The former was indicated by the disturbed response to sheep erythrocytes injections, by the decreased number of spontaneous E rosette forming lymphocytes, the prolonged survival of skin allografts and the significant inhibition of the delayed hypersensibility skin reaction (tuberculination) following administration of Mycobacterium microti. The latter was based on the disturbed response to a subcutaneous dose of tetanus toxoid. The reaction of lymphocytes of ABLS treated calves to phytohemagglutinin and poke weed mitogen was also inhibited. The disturbed reactions of the T and B cells might be among others based on the strong reduction of lymphocytes in the blood circulation by ABLS (up to 10–20%).

Suggestions with regard to further applications and studies with ABLS were given.