Cell Separation on Affinity Columns: The Isolation of Immunospecific Precursor Cells from Unimmunized Mice

Abstract

We have used affinity columns to isolate from the spleens of unimmunized mice a population of lymphocytes that specifically bind a lactoside hapten. These cells are able to initiate an antibody response to azophenyl-j3-lactoside when transferred to appropriate irradiated recipients.Considerable evidence supports a clonal selection theory of antibody formation (1). It is generally believed that normal, unimmunized vertebrates contain many diverse populations of lymphocytes that exhibit antibody-like receptors on their surfaces and that a given antigen stimulates only selected precursor cells to proliferate and to give rise to antibody-producing cells of corresponding specificity. For secondary lymphoid tissues, estimates of the frequency of precursor cells specific for a multideterminant antigen (e.g., foreign erythrocytes or polymeric flagellin) are in the range of 5 X 10-4 to 10-5 (2). To investigate cellular differentiation in the immune system, it should be advantageous to obtain pure populations of lymphocytes with a common receptor specificity that can be induced to antibody response or tolerance to a defined antigenic determinant.We report here the specific purification of precursor cells for a primary antibody response to azophenyl-#-lactoside (N2Phlac). The cells have been isolated from the spleens of unimmunized mice by filtration of cell suspensions through affinity columns of large polyacrylamide beads to which N2Ph-lac groups are covalently attached (3, 4 MATERIALS AND METHODSPreparation of N2Phlac-Affinity Beads. N2Phlac-affinity beads were prepared as before (3), except that the time of reaction of the Bio-Gel with hydrazine was reduced to 1.25 hr to limit the number of amide groups modified (6) and to better preserve the structural integrity of the beads. Mechanical stirring was avoided in all preparative procedures.Specific Cell Purification. Single-cell suspensions were prepared from pooled spleens of 4-to 6-months-old unimmunized Balb/c mice. Cells were washed and resuspended in balanced salt solution (BSS) (7) without glucose immediately before addition to columns. Column procedures followed those described for column preparation of pure populations of antiN2Phlac-specific lymphocytes (4). In the present experiments, we used several small columns, each containing 2.5 ml of N2Phlac-affinity beads. About 5 X 108 cells, at a concentration of 8 X 108 cells per ml, were passed through each column, which was then washed with 5 ml of BSS. Columns were then equilibrated with buffer (pH 7.2) containing p-aminophenyl-3-lactoside; cells were eluted from each column in a 4 ml volume of BSS, 15 IM in the lactoside hapten, and containing 0.1% glucose. When the cells were to be used in transfer experiments, irradiated (1500 R) normal thymocytes were added to the eluting buffer (pH 7.2) at a concentration of 107 cells per ml. This was necessary so that the specifically eluted cells could be recovered efficiently during two centrifugation cycles required for concentrating the cells and diluting out th...

The Mechanism of Antigenic Stimulation of Primary and Secondary Clonal Precursor Cells

“…The Nature of Carrier-Specific Amplification of Slimulation.--Irradiated, carrier-primed recipient animals and cells from such animals have been used previou'sly to enhance both primary and secondary responses (14,(30)(31)(32)(33)(34)(35). The experiments reported here utilized the transfer of cells to carrier-primed, irradiated recipient mice to permit an analysis of both primary and secondary antigenic stimulation of clonal precursor cells specific for a haptenic determinant.…”

“…While it is clear that precursor cells specifically bind antigen (11, 13,33,42,43), the act of binding antigen is not a sufficient condition for stimulation of these cells. This is best exemplified by the fact that at high concentrations both free haptenic determinants and hapten on heterologous carriers bind receptors so as to inhibit stimulation, but do not stimulate antibody production.…”

Immunological Function of Lymphocytes Fractionated with Antigen-Derivatized Fibers

“…These include bead columns (1,2), fluorescence-activated cell sorting (3), and differential sedimentation of rosettes (4). These fractionation systems are not entirely adequate, however, either because they do not yield specific antigen-binding cells of both the T and B classes, because they yield cells that are altered in their response to antigen, or because they yield cell populations that are only partially enriched with respect to their antigen-binding specificity.…”

Cell Fractionation on Affinity Columns

“…This is particularly pertinent to the interpretation of antigen-binding in normal but potentially responsive animals when affini ty chromatography, which depends on this binding, is employed to delete from cell populations the very small number of antigen-binding cells pre sent. There is increasing indirect evidence for the functional role of such cells, based on the deletion of specific humoral responsiveness from cell populations by such methods [19], but less extensive direct evidence for the positive participation of such cells in a specific response [6],…”

“…In nonimmunized but potentially responsive animals, M oro7./Kotoulas such methods have permitted the deletion of cells responsible for specific responses and the demonstration of their antigen-binding activity and spe cific proliferation [reviewed in reference 19]. However, direct evidence for enhancement of a specific immune response by such 'virgin' lympho cytes is limited [1,6],…”