Immunological memory in mice. I. Physical separation and partial characterization of memory cells for different immunoglobulin classes from each other and from antibody-producing cells

Plaque forming cells (PFC) of different immunoglobulin classes producing antibodies against sheep erythrocytes were separated according to their buoyant densities by means of equilibrium centrifugation in a stepwise BSA gradient. In the period of 7–10 days after immunization γM PFC are markedly enriched in fractions of low density and relatively depleted in fractions of high density. The distribution of total γG PFC shows less enrichment in the lower density fractions and less depletion in the higher density fractions. The density profile for γG_2a PFC is even flatter, with a significant difference (depletion) relative to the unseparated spleen cells only in the highest density fraction. The density gradient distributions of cells able to transfer an adoptive immune response of the various immunoglobulin classes are markedly different from the PFC distribution. Cells obtained 7–10 days after immunization able to transfer an IgM response are present in the same proportions across the density gradient, whereas memory cells for γG_2a obtained at this time are markedly enriched in fractions of low density and virtually depleted from high density fractions. With increasing time after primary immunization, the γG_2a memory cells increase progressively in density and by 6 weeks the higher and lower density fractions have the same proportions of γG_2a memory cells. The total γG (mainly γG₁) memory cells by 7–10 days show slight enrichment in low density fractions and no depletion in high density fractions. The conclusions were reached that (a) memory for γG₁ develops earlier than memory for γG_2a and (b) that memory for anti-SRBC antibodies of different classes is carried in separate cells. When gradient fractions enriched for PFC and memory cells for all classes were completely depleted of PFC using glass bead columns, the ability of this fraction to transfer memory for all classes was not diminished. This shows that memory cells are not identical with cells secreting antibodies.