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.

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.

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.

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.

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.

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.

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.