There is conflicting evidence about the relative roles of division and "recruitment" of responding ceils during the early primary immune response. The cells primarily releasing "19S" hemolysin for sheep erythrocytes may be enumerated using the plaque-forming cell technique developed by Jeme and Nordin (1, 2) and independently by Ingraham (3). An exponential increase in plaque-forming cells occurs during the early primary response; doubling times vary from about 5 to 10 hr depending on the dose of antigen and other experimental variables (4-14).A method using mitotic blocking agents to estimate cell cycle times of antibody-formlng cells, in vivo and in vitro, is presented. Results with this method indicate that the increase in numbers of antibody-forming cells which occurs during the early primary response is due primarily to exponential division of responding cells. Increased responses produced by larger antigen doses or adjuvant appear to result from an increased rate of division of responding cells. These findings are contrary to the view that the observed increase in antibody-producing ceils can be attributed to continued activation or recruitment of potential antibody-producing cells (9-14). Materials and MethodsYoung adult Sprague Dawley rats were used. The antigen was washed whole erythrocytes prepared from sterile sheep's blood. A single sample of blood stored in Alsever's solution was used in each experiment both for ;mmunization and for titrating immune respo~es. The sheep erythroc~es were washed four times in large volumes of freshly prepared pyrogenfree saline and used immediately. Doses of sheep erythrocytes were standardized by ceil *

The suppressive effects of monospecific goat anti-mouse globulins on primary immunoglobulin class-specific plaque-forming cell responses in mouse spleen cell cultures were investigated. Anti-µ suppressed responses in all immunoglobulin classes, whereas anti-γ1 and anti-γ2 suppressed the γ1 and γ2 responses but not γM or γA responses, and anti-γA suppressed only γA responses. The mechanism of action of the anti-µ was studied in detail because of its suppression of responses in all immunoglobulin classes. The anti-µ was specific for µ-chain determinants; its activity was dose dependent, but was not mediated by killing cells with surface µ-chain determinants. Free γM but not γG myeloma proteins in solution effectively competed with µ-bearing cells for the anti-µ. An excess of anti-µ was necessary in the cultures for 48 hr to insure complete suppression of 5-day responses. However, after removal of excess anti-µ at 48 hr, responses could be stimulated by newly added antigen in cultures where incubation was prolonged to 7 days. Anti-µ was most effective when added at the initiation of cultures and had no suppressive effect when added at 48 hr. Excess antigen did not effectively compete with anti-µ for antigen receptors. Precursors of antibody-forming cells were shown to be the cell population where the suppressive activity of anti-µ was mediated. The experiments suggest that anti-µ combines with µ-chain determinants in antigen-specific receptors on the surfaces of antibody-forming cell precursors, prevents effective stimulation by antigen and subsequent antibody production. To explain suppression of responses in all Ig classes by anti-µ, several models were proposed. It is not possible to determine from the data whether stimulation of precursor cells with γG or γA receptors requires concommitant stimulation of separate cells with only γM receptors, or whether cells bearing γM receptors are precommitted to or differentiate into cells capable of synthesis of other Ig classes, or whether receptors of γM and another Ig class are present on some virgin precursors or the second Ig receptor appears after antigenic stimulation.

Precursors of antibody-producing cells in the mouse are functional homologues of bursa-derived lymphocytes in birds (1); they have antibody-like immunoglobulin receptors (2-8) specific for limited antigenic moieties (6-16) on their cell membrane which are largely of the 3,M immunoglobulin (Ig) 1 class in experimentally virgin animals of this species (16,17). Combination of antigen with the specific receptor and the cooperation of specific thymus-derived lymphocytes triggers division and differentiation of these precursor cells into mature antibody-secreting cells (6,(18)(19)(20)(21)(22)(23).In the preceding paper (24), we demonstrated that antibody to mouse /z-chain specifically suppressed primary plaque-forming cell (PFC) responses of all Ig classes in mouse spleen cell cultures stimulated with heterologous erythrocytes. This suppressive effect was mediated through the precursors of antibody-producing cells and appeared to involve, for the most part, a potentially reversible saturation of antigen receptors with/z-chain determinants by the anti-/z and prevention of antigenic stimulation rather than elimination of these virgin precursor cells with surface /z-chains via cell death. Antibodies to mouse 3`1 and 3'2 heavy chains suppressed both 71 and 3'2, but not 3"M or 3`A primary PFC responses.