A comparative analysis of the mitogenic response of intestinal intraepithelial lymphocytes in various age groups of turkeys.

Mitogenic responsiveness of intestinal intraepithelial lymphocytes (i-IEL) to concanavalin A (Con A), phytohemagglutinin P (PHA-P), and lipopolysaccharide (LPS) from Salmonella typhimurium were evaluated in various age groups of turkeys by a colorimetric blastogenic microassay. Comparisons were made between mitogenic responses of turkey i-IEL and peripheral blood lymphocytes (PBL). The results from this study demonstrated that i-IEL and PBL of turkeys responded to T-cell mitogens, Con A and PHA-P, in every age group examined. The LPS induced a significant mitogenic response in PBL but not in i-IEL of turkeys. The mitogenic responses of turkey i-IEL and PBL to the three mitogens examined were similar to mitogenic responses observed in an earlier study performed by using chicken i-IEL and PBL. The results indicated a difference in mitogenic response between different age groups. An increase was found in mitogenic response of i-IEL to both T-cell mitogens from 3 days of age to 1 wk of age, whereas mitogenic response of PBL to all three mitogens declined significantly from 1 day of age to 3 days of age. The highest mitogenic response of i-IEL to T-cell mitogens was observed at 1 wk of age. The highest mitogenic response of PBL to both T-cell mitogens was observed at 1 day of age and the highest PBL response to LPS was observed at 16 wk of age. The mitogenic response induced by PHA-P provided less variability between age groups than the mitogenic response induced by Con A.     

Frequency of lymphocytes bearing Fc receptors and surface membrane immunoglobulins in normal, persistent lymphocytotic and leukemia cows.

Fluoresceinated, heat-aggregated bovine immunoglobulins (B-IgG) and human immunoglobulins (H-IgG) were used to detect a receptor for the crystallizable fragment (Fc) of the immunoglobulin molecule on peripheral blood lymphocytes (PBL) of cattle. The aggregated and B-IgG and H-IgG bound to the bovine PBL, but aggregated H-IgG was found to be more sensitive for the detection of Fc receptors. The specificity of aggregated H-IgG binding to the Fc receptors was established by demonstrating that antigen-antibody complexes inhibited this binding, and unaggregated H-IgG did not bind significantly to PBL. Double-labeling experiments suggested that all Fc+ cells have surface immunoglobulins (SIg), a marker for B lymphocytes. The percentage of Fc+ and SIg+ cells in normal animals was 9.5% (range 4-15%) and 16.2% (range 4.5-30.2%), respectively. Persistent lymphocytotic cows had 2.71 times more Fc+ and 3.85 times more SIg+ lymphocytes than did normal cows. Cows with lymphosarcoma had a lower percentage of Fc+ and SIg+ cells than did cows with persistent lymphocytosis. Cases with thymic lymphosarcoma and those with the skin form of leukemia had normal percentages of Fc+ and SIg+ cells.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP)

Untreated and neuraminidase-treated equine peripheral blood lymphocytes were analysed for binding of the A hemagglutinin of the snail Helix pomatia (HP). For optimal staining by direct immunofluorescence, the concentration of neuraminidase had to be increased as compared to that needed for other species. Moreover, higher concentrations of HP were required for optimal staining of equine lymphocytes as compared to lymphocytes from other species. Even so, the maximal number of equine lymphocytes exhibiting positive staining was only about 20%. No, or very few, HP-positive lymphocytes were seen when neuraminidase treatment was omitted. However, when the more sensitive method of indirect immunofluorescence was used, approximately 60% of the lymphocytes were HP positive without prior treatment with this enzyme. Neuraminidase treatment significantly increased this figure to about 75%. In all instances, HP binding was specific since it was inhibited by the competitive sugar hapten N-acetyl-D-galactosamine (D-GalNAc) while addition of D-glucose (D-Glc) gave no inhibition. HP binding to neuraminidase-treated lymphocytes was also investigated quantitatively by means of 125I-labeled HP. The number of HP molecules bound per HP-positive cell was approximately 3 X 10(5) and the apparent association constant for the binding of HP to its cellular receptors was approximately 8 X 10(7) 1/mol. No binding of HP to untreated lymphocytes could be recorded in these experiments.     

Duck lymphocytes. VI. Requirement for phagocytic and adherent cells in lymphocyte transformation.

Preparations of duck (Anas platyrhynchos) spleen and blood lymphocytes depleted of cells capable of phagocytosing carbonyl iron gave lower transformation responses to the mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), lentil lectin (LL) and phorbol ester (PMA) than intact cell preparations. When cell populations were fractionated on the basis of their adherence to plastic, it was found that the adherent cells were responsive to PHA, Con A, BSS, WGA and PMA, while the non-adherent cells responded to LL. These observations confirm the expected requirement for phagocytic accessory cells in the induction of in vitro mitogen-driven duck lymphocyte responses. The responses of plastic-adherent populations of cells to most mitogens are believed to reflect the generally close physical relationship between the adherent accessory cells and the lymphocytes, although it remains possible that duck monocytes respond to some of the mitogens employed. The data also suggest that LL stimulates a population of cells different to those responding to other mitogens.     

T and B peripheral blood lymphocytes in normal and lymphocytotic sheep

Surface immunoglobulins (SIg), Peanut Agglutinin (PNA), spontaneous erythrocyte rosette (E-rosette) and Helix pomatia (HP) marker were investigated in normal and Bovine leukemia virus (BLV)-infected sheep. In normal sheep, 19.3% +/- 4.9 of peripheral blood lymphocytes (PBL) were SIg+, whereas 58% +/- 5.69 were PNA+, and 19.6 +/- 5.2 were E-rosette forming cells (E-RFC). In BLV-induced lymphocytotic sheep, SIg+ cells in PBL reached 59.4% +/- 15.06. In the same animals, PNA bound to 20.6% +/- 9.69 and E-RFC were 8.7% +/- 4.5. A panning technique was applied with an anti sheep-immunoglobulins coated plates to separate SIg+ (adherent cells = A) and SIg- cells (non-adherent cells = NA). The (A) population was 94-95% SIg+ cells and 2-3% PNA+, while the (NA) population was 0-4% SIg+ and 79-85% PNA+ cells. Thus PNA is a T cell marker in sheep species. HP, a marker for bovine T lymphocytes was also studied. Sheep PBL do not bind to HP. However, after panning separation about 50% of NA cells became HP+.     

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%).     

B and T lymphocytes in the bovine mammary gland: Rosette formation and mitogen response

Characterization of lymphocyte subpopulations in the bovine mammary gland was accomplished using cells obtained from dry secretions. Correlation of cell surface properties with functional capacity was attempted by assaying the ability to form erythrocyte-antibody (EA) rosettes, erythrocyte-antibody-complement (EAC) rosettes, and sheep erythrocyte (E) rosettes and the ability to respond to phytohemagglutinin (PHA), Concanavalin A (Con A), and pokeweed mitogen (PWM) in the lymphocyte stimulation test. Results were compared with those obtained for peripheral blood lymphocytes (PBL) from the same animals. Mammary gland lymphocytes (MGL) formed significantly fewer (p < .01) EA and EAC rosettes, but significantly greater (p < .01) E rosettes compared to PBL. MGL were significantly less responsive (p < .05) to mitogens than were PBL. MGL contained a large proportion of T lymphocytes, which do not respond to T lymphocyte mitogens in culture.     

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.     

Production of interleukin 2 and expression of interleukin 2 receptors by pony peripheral blood lymphocytes after stimulation with a soluble fraction of Trypanosoma evansi

Pony peripheral blood lymphocytes (PBL) were stimulated with a soluble fraction of Trypanosoma (T.) evansi (SF). As determined by 3H-thymidine incorporation, the cells underwent a proliferative response and were able to: a) produce a factor having the biological activities of interleukin 2 (IL-2) since their supernatants could support the in vitro growth of pony PBL stimulated with concanavalin A (Con A-blasts); b) undergo a further proliferative response when incubated in short term cultures with SF, human recombinant IL-2 (hrIL-2), or both c) bind specifically radiolabelled hrIL-2 (125I-hrIL-2). The date described here indicate that a soluble fraction of T. evansi stimulated pony PBL which subsequently produced IL-2 and expressed IL-2 receptors (IL-2R).     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Effect of infections with swine fever virus on immune functions II. Lymphocyte response to mitogens and enumeration of lymphocyte subpopulations

Peripheral blood and spleen lymphocytes from pigs infected with a low-virulent strain of swine fever virus (SFV) were transiently hyporesponsive to the mitogenic action of PHA, PWM and Con A. The mitogenic reactivity of lymphocytes from lymph nodes from such pigs appeared to be enhanced rather than depressed at that time. In addition, hyperresponsiveness of peripheral blood lymphocytes (PBL) to these mitogens occurred in some pigs.PBL from pigs lethally infected with virulent SFV showed a persistent depression of the response to these mitogens, whereas lymphocytes from lymph nodes had a high responding capacity.A lymphocyte response to SFV antigens could not be demonstrated in infected pigs.These SFV infections did not markedly affect the percentage of lymphocytes in the blood and most lymphoid organs rosetting with sheep red blood cells. On the other hand, surface immunoglobulin-bearing lymphocytes were markedly increased in lymph nodes from pigs exposed to virulent SFV. The sum of both lymphocyte subpopulations in the lymph nodes from these pigs often considerably exceeded the 100% value, which strongly suggests the presence of cells bearing both surface immunoglobulin and receptors for dextran-treated sheep red blood cells.Possible correlations between these findings are discussed. The results suggest that infections with SFV induce systemic alterations in the process of lymphocyte recirculation in the pig.     

The distributions of phytohemagglutinin-P and concanavalin A binding sites on equine, bovine and canine peripheral blood lymphocytes

The distributions of phytohemagglutinin-P (PHA) and concanavalin A (ConA) binding sites were investigated for equine, bovine and canine peripheral blood lymphocytes (PBL). Non-B lymphocytes were collected from each PBL using a fluorescence-activated cell sorter (FACS), and the numbers of PHA and ConA binding sites on their surfaces were counted. Most PHA binding sites on PBL of the three species were shown on the surfaces of non-B lymphocytes. On the other hand, the ConA binding sites on equine and canine PBL existed mainly on the surfaces of non-B lymphocytes, but B lymphocytes of these two species had many ConA binding sites. These results were confirmed by the results of two-parameter fluorescence analysis using FACS. It is, therefore, concluded that the different optimum concentrations of PHA and ConA in PBL blastogenic responses of each animal depended on the different distributions of their binding sites.     

In vitro modulating effects of porcine immunoglobulin G on mitogens-induced lymphocyte response in precolostral, suckling and weaned piglets

The influence of allogeneic IgG on in vitro reactivity of peripheral blood lymphocytes (PBL) of neonatal colostrum-deprived piglets as well as of suckling and weaned piglets was studied. PBL were preincubated with purified allogeneic IgG for 24 h before their ability to respond to PHA, Con A or PWM was tested. PBL of precolostral piglets pretreated with allogeneic IgG exhibited higher response to PHA (P less than 0.01) than untreated control cells. An increased response of PBL treated with IgG was also observed in suckling piglets as compared to their respective control cells (P less than 0.01). Responsiveness of PBL treated with IgG to PWM was suppressed. No differences in response to Con A regardless of the sources of lymphocytes was observed as compared to IgG untreated controls. The results suggest that pretreatment of lymphocytes of piglets with allogeneic IgG modulates their reactivity to mitogens, suppressing the response to PWM and stimulating the response to PHA, respectively.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

In vitro lymphocyte stimulation by concanavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis.

Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentrations of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indices neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37 degrees C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubation in cell-culture medium between normal and atopic dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type 1 hypersensitivities such as atopy, can modulate lymphocyte function. At 10(-4) and 10(-8) M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H1 and H2 antagonists, we concluded that histamine enhanced mitogenesis via H1, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.     

Interleukin 2 (IL-2) production by mitogen stimulated bovine peripheral blood lymphocytes and its assay

Culture medium from bovine peripheral blood mononuclear cells stimulated with the mitogens phytohaemagglutinin (PHA) or Concanavalin A (Con A) was found to maintain the proliferation of Con A blasts in vitro. The factor responsible for this activity was not absorbable with bovine erythrocytes or fresh peripheral blood lymphocytes but was removed by Con A blasts. Production of this factor was dependent on the dose of mitogen used and was greatest after 24 h culture compared to 48 h. Quantitative determinations of factor activity in supernatants were carried out by regression analysis of logit transformed data from assays measuring the maintenance of Con A blast proliferation by supernatants.     

Canine transmissible venereal tumor cell depletion of B lymphocytes: molecule(s) specifically toxic for B cells

Canine transmissible venereal tumor (CTVT) is an excellent model for investigating the interaction between host immunity and tumor growth. Although CTVT is an allograft, initially the host immune system is unable to destroy the tumor cells, and the tumor grows progressively for about 4-6 months (P phase). After a short stable phase, the tumor undergoes regression (R phase). In this study, CTVT inoculation significantly reduced the proportion of B lymphocytes among all peripheral blood lymphocytes (PBL), but the proportion of B lymphocytes returned to normal after complete removal of CTVT. Following CTVT inoculation, immunoglobulin concentrations decreased gradually, coincident with B lymphocyte decline. Furthermore, CTVT secreted a soluble, heat- and protease K-sensitive cytotoxic molecule(s) that destroyed peripheral blood B lymphocytes (PBBL) but spared other types of immune cells regardless of whether mitogens, such as IL-2 or Con A, were present. The decrease in the proportion and viability of PBBL was caused by a cytotoxic molecule(s) that induced apoptosis. The molecular weight of the CTVT-derived cytotoxic molecule(s) was 30-100kDa. Human, domestic cat, horse and mouse B cells were also sensitive to the substance.     

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.     

Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes

The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied. Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2. The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action. Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM. The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM. The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum. When proliferation kinetics were studied, the peak response was observed on Day 2.