Concanavalin A-induced electrokinetic changes and redistribution of receptors in mouse thymocytes.

Thymocytes are known to consist of mature and immature cells. The assessment of Con A-induced changes in electrophoretic mobilities (EPM) and receptor redistribution in thymocytes of AKR mice revealed two distinct sub-populations of cells. Sub-population A showed receptor redistribution and increase in EPM at low concentrations of Con A (5 micrograms/ml). Majority of these cells showed two sets of sequentially reacting receptor sites for the lectin. Cells belonging to sub-population B, in contrast, required higher concentrations (15-25 micrograms/ml) of Con A for the redistribution of receptors. Second set of receptors, reacting with Con A after the redistribution of the first set, could not be detected on these cells. High concentrations of Con A inhibited receptor mobility on all the thymocytes. The receptor redistributional profile of cells in the sub-population A was similar to that of mature splenic T-cells while cells in sub-population B resembled the immature leukaemic cells in this respect. These investigations provide an additional parameter to study cellular heterogeneity in thymus.

Lactic dehydrogenase isozyme distributions in lymphocytes from normal and leukemic mice.

LDH isozyme distributions were studied in the thymocytes of normal and spontaneously leukemic mice. In the normal animals, H:M ratios were found to be higher for more mature cells of adult animals than for less mature thymocytes of the neonates. However, thymocytes from leukemic animals bearing mature phenotype displayed very low values of H:M ratios. From these results, the relationship between the changes in LDH-isozyme distributions in AKR thymocytes and their progressive maturation appears to be equivocal. Alterations in isozyme distribution patterns, reflecting a decrease in H:M ratios, on the other hand, appears to be a characteristic feature of terminal stages of this murine leukemia.

Suppression of skin allograft rejection by post-transplantation administration of specific anti-lymphocyte serum.

An heterologous anti-lymphocyte serum ALS(I-GR), was raised in rabbits by immunization with draining lymph node cells of AKR mice which had rejected DBA/2 skin allografts. Treatment of AKR mice with this ALS on the 4th day after DBA/2 skin grafting, significantly prolonged the survival of the graft in comparison with that in allografted mice treated with normal rabbit serum. In contrast, ALS prepared against unsensitized lymph node cells was found to be ineffective when administered after transplantation. A further prolongation of allograft survival was obtained when ALS(I-GR) was administered to recipients on days +4 and +7. ALS(I-GR) seemed to specifically suppress the rejection of DBA/2, but not of C57 BL/6 skin grafts. The suppressive action of ALS(I-GR) was not due to cross reactive (anti-DBA) antibodies and was probably directed against idiotypic determinants on antigen-stimulated cells.

Electrokinetic status of the subpopulations of lymph node cells in contact-sensitized CBA mice.

Electrokinetic behaviour of LNC and their subpopulations from CBA mice contact-sensitized with dinitrofluorobenzene and oxazolone were studied. The sensitized LNC showed a significant reduction of mean EPM on the days of maximum DH response. Histogram analysis revealed that LNC of unsensitized as well as contact-sensitized mice were heterogeneous populations. While the LNC of unsensitized mice resolved into two subpopulations, sensitized LNC resolved into three distinct subpopulations. The emergence of a new populations with mean EPM intermediate between those of low and high mobility lymphocytes was a consequence of contact sensitization. Enriched subpopulations were also obtained by nylon adherence-dependent and surface marker-dependent procedures. histograms of these subpopulations revealed that in the mice sensitized to DNFB and oxazolone both T and B cells were electrokinetically altered and were heterogeneous in the distribution of their EPM. These findings are similar to the earlier observations on EPM of LNC in allograft-sensitized mice.

Niridazole-mediated modulation of suppressor cells in Wistar rats.

The antischistosomal drug niridazole has been shown to inhibit inductive (Vadas and Bernard, 1981) as well as effector phases of delayed hypersensitivity (Sainis et al., 1983). Furthermore, it also abrogates help for delayed hypersensitivity in antigen-primed animals (Sainis et al., 1983). The effect of this drug on antigen-induced suppression was examined in the present studies. Profound suppression of delayed hypersensitivity to sheep erythrocytes was obtained in Wistar rats given 10(8) erythrocytes (i.v.) 6 days before the immunizing dose (2 x 10(9) erythrocytes, i.p.). When these rats were orally administered niridazole (50 mg/kg) 7 days before the tolerising dose of antigen, suppression of delayed hypersensitivity was not obtained. Splenic lymphocytes of rats given the tolerising dose 6 days earlier adoptively transferred the suppression to inbred recipients. Treatment of these afferent suppressor cells with sera from niridazole-treated unimmunized rats abrogated their function. Likewise, the efferent suppressor cells obtained from fully tolerised rats did not suppress the delayed hypersensitivity when co-transferred with immune lymphocytes, if they were pretreated with niridazole-active serum. The metabolite of niridazole present in this serum seems to impair the suppressor cells functionally. Niridazole may thus prove to be a versatile immunomodulator for effector, helper and suppressor T-cells.

The subpopulations of lymph node cells from allograft-sensitized AKR mice.

Electrokinetic behaviour of skin allograft-sensitized LNC and their subpopulations from AKR mice were studied. Sensitized LNC showed a significant reduction in the mean EPM in the post-transplantation period. Histogram analysis of both unsensitized and sensitized LNC of AKR mice showed a heterogeneous population. This heterogeneity in the lymph node cells was more pronounced in the case of sensitized cells. While unsensitized cells resolved into two subpopulations, sensitized LNC resolved into three distinct subpopulations. The emergence of a new population in the case of sensitized cells may be a consequence of allograft sensitization. Subpopulations of unsensitized and sensitized LNC were also obtained by surface marker dependent and differential adherence to nylon wool procedures. Histograms of these subpopulations showed heterogeneity, indicating that both T and B lymphocytes are affected by allograft sensitization. These findings were further confirmed by interacting the subpopulations with specific HCA antigen.

Karyological studies on established mosquito cell lines.

Chromosome frequency distribution and cellular DNA estimations in different established mosquito cell lines were studied. These cell lines exhibited a wide range of cell types with a diploid stem-line comprising 50-55% and a haploid substem-line comprising 12-30% of the population. Estimation of cellular DNA contents by impulse cytoflowmetry and by Feulgen cytophotometry supported these observations. Because of their low diploid counts, these cell lines cannot be classified as diploid.

Contact sensitivity to dinitrofluorobenzene and oxazolone in mice: in vitro cell-electrophoretic assay.

Prevalent techniques for the assessment of cell-mediated immunity (CMI) in vitro are time consuming and may lack specificity. Deriving from the earlier observation of reduction in mean electrophoretic mobility (EPM) of lymphocytes following the development of humoral immune response, the possibility of using this electrokinetic method for the assessment of contact sensitivity in vitro was tested. CBA mice epicutaneously sensitized to dinitrofluorobenzene and 2-phenyl 4-ethoxy oxazolone showed a marked and reproducible reduction in the mean EPM of their lymph node cells (LNC). The specificity of this alteration in surface charge density was established by the enhancement in the EPM of contact sensitized LNC upon subsequent incubation in vitro with the specific antigen only. The profiles of time kinetics of changes in the EPM of LNC before and after incubation with antigen were virtually parallel to those of in vivo delayed hypersensitivity (DH) reaction as measured by the ear-swelling method. The coefficients of correlation between the reduction in EPM and in vivo DH response for DNFB and oxazolone were 0.89 and 0.86 respectively. EPM measurements may thus provide a sensitive, rapid, and quantitative parameter for the assessment of CMI in vitro.

Mechanism of specific suppression of delayed hypersensitivity response by anti-lymphocyte serum.

Mechanism of antigen-specific immunosuppression exhibited by a heterologous anti-rat lymphocyte serum, ALS (CM), was investigated. This ALS possessed antibody activity against T-effector cells responsible for the expression of delayed hypersensitivity (DH) to sheep erythrocytes (SRBC) in rats. Immune lymphocytes were treated in vitro with ALS (CMI) and transferred to inbred recipients. ALS (CMI) inhibited the ability of the immune cells to transfer DH. Prior absorption of ALS (CMI) with gamma-globulin from rat anti-SRBC serum or the immune cells abolished this suppressive ability. These results demonstrated that ALS (CMI) had anti-idiotype activity.

The status of concanavalin A receptors on the lymphocytes of leukemic AKR mice: inhibition of redistribution by high concentrations of the lectin.

Cell-electrophoretic and fluorescence microscopic investigations were carried out on the interaction of concanavalin A with the splenic lymphocytes of normal and spontaneously leukemic AKR mice. The normal splenic lymphocytes (NSL) showed a biphasic profile of electrophoretic mobility (EPM) as a function of the concentration of Con A, under capping conditions. The mean EPM of NSL increased at low concentrations of the lectin and was reduced below that of untreated cells at higher (greater than or equal to 15 micrograms/ml) concentrations of the lectin. The leukemic cells (LSL) also showed enhancement in EPM at low concentrations of Con A. At high concentrations of the same, however, the mean EPM of LSL was the same as that of untreated cells. In the case of NSL the reduction in EPM at high concentration of Con A is known to be brought about by post-redistributional binding of excess lectin to new receptor sites which emerge after capping of the first type of receptors. Similar investigations of the electrokinetic characteristics of Con A-receptor interaction on leukemic cells revealed that only a single type of receptor was present on LSL. These receptors were inducible to redistribution at low concentrations of Con A. High concentrations of the lectin, however, inhibited the redistribution of the receptors to Con A on LSL. This was confirmed when LSL treated with high concentrations of Con A were relieved from this inhibition by moderate concentrations of alpha-methyl glucoside, which dissociates cell bound Con A. Very low or very high concentrations of alpha-MG were ineffective. The receptors to Con A on LSL were thus behaviorally distinguishable from those on NSL. These data clearly demonstrate that the malignant transformation in AKR mice is also associated with alterations in the properties of receptors to a multivalent ligand Con A.

Quantitative assay for lectin-induced cytoagglutination by means of an electronic particle-counting technique.

A method employing an electronic particle-counting technique was used to quantify lectin-induced agglutination of human granulocytes and lymphocytes with either concanavalin A or wheat germ agglutinin. The number and mean volume of single cells and aggregates in the presence of increasing concentrations of lectin were computed from 95% confidence intervals. Agglutination depended on both the number of free cells and the number and size of the cell clusters. Changes in these two variables were mutually independent of one another, and both were simultaneously determined. An index of agglutination that takes the effect of these two variables into account was defined as (formula: see text) VA equals mean volume of cell aggregates, NS equals number of single cells, NA equals number of cell aggregates, VS equals mean volume of single cells, rb equals r at a given lectin concentration, and ra equals r in the absence of lectin. For any combination of lectin and cell type, the agglutination curve, as described by zeta, consisted of two components: a) a flat region in which zeta remained constant with increasing lectin concentrations and b) a region in which zeta increased linearly as a function of the logarithm of lectin concentration. The shapes of these curves offered two parameters for quantitative comparison of agglutinability: 1)threshold concentration, defined as the minimum concentration of lectin (microgram/ml) required to bring about a measurable rise in zeta and 2) the concentration gradient, defined as the change in zeta for an increase of one log unit in the concentration of the lectin in the range beyond the threshold concentration. This method offers a high degree of quantification and provides reliable information that can be meaningfully correlated with cell surface characteristics.

Relationship between concanavalin A-induced agglutinability of murine leukemia cells and their propensity to form heterotypic aggregates with syngeneic lymphoid cells.

The agglutinabilities of the murine leukemia cell lines L1210, P388, and C1498 were determined in the presence of concanavalin A (Con A) by a quantitative cytoagglutination assay. The propensity of these cells to form heterotypic aggregates with normal syngeneic spleen cells, those obtained from mice carrying the respective leukemias in ascitic form, and syngeneic lung cells also were determined. Con A caused agglutination of all five types of leukemia cells and the resulting agglutination patterns had certain characteristics. Threshold concentrations of Con A, below which no significant cytoagglutination occurred, were very low. A steady increase in zeta, a previously defined index of agglutination that simultaneously takes into account the number of free cells and the number and size of the aggregates, was observed with increasing concentrations of Con A until a plateau was reached at 25-50 microgram/ml and this extended over a wide range of lectin concentrations (50-1,000 micrograms/ml). Self-aggregation of leukemia cells was not observed, and their propensity to form heterotypic aggregates with syngeneic lung cells was negligible. However, all leukemia cell lines formed measurable aggregates with spleen cells from both normal and leukemia-bearing mice; these aggregates usually reached a maximum plateau between 30 and 35 minutes of incubation and remained constant thereafter. Aggregation of leukemia cells with spleen cells from leukemic mice always was greater than that with spleen cells from normal mice. Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A receptor carbohydrate moieties may be involved in the intercellular adhesion leading to heterotypic aggregate formation.

A sub-set of murine T lymphocytes forming rosettes with rabbit erythrocytes.

Lymphocytes from two murine strains AKR and CBA were found to form rosettes with rabbit erythrocytes. On comprehensive characterization of these cells by enrichment or selective depletion of constituent T- or B-lymphocytes, rosette forming ability was found to be an attribute of the subpopulation of Thy-1 +ve and Ig -ve cells. Further, the lymphocytes of this subset of T cells were found to be devoid of the receptor for C3 and a majority of them displayed sensitivity to hydrocortisone.

Confirmatory evidence for the existence of two types of receptor sites for Con A on mouse lymphocytes.

Splenic lymphocytes of normal AKR mice (NSL) were sequentially treated with two Con A preparations, each labelled with a different fluorochrome. cells were allowed to cap rhodamine-labelled Con A (TRITC-Con A). They were subsequently incubated with fluorescein-labelled Con A (FITC-Con A). FITC-Con A bound to cell in the region of the cell membrane which was free of the first label after capping. Such mutually exclusive localization of Con A receptors was not observed in the NSL which did not undergo redistribution. This provides direct confirmatory evidence in support of the existence of two behaviourally distinct types of Con A receptors on AKR lymphocytes.