Various theories of antibody formation (1,2,3) propose that the primary antigenic stimulus is mediated by preformed specific "natural" antibody molecules.A large amount of data has now accumulated (4--10) indicating that passively administered specific antibody has a depressive effect on the immune response. Both preparations of 7S antibody and, to a lesser degree, preparations of 19S antibody were found to be depressive. It has been proposed (4) that the antibody acts directly on antigen-sensitive target ceils, but the evidence now suggests that antibody inhibits the response by combining with antigenic determinants. Recently, it has been found (11) that when two hapteus are present on one carrier molecule, suppression of antibody formation against one determinant does not necessarily suppress antibody formation against the second determinant. It therefore appears that suppression of the response by antibody does not imply a prevention of the antigen from reaching the target area. Possibly, binding of antigenic determinants by antibody reduces the number of free determinants available for stimulation.Though apparently contradicted by these findings, the theories alluded to in the first paragraph, do not lack experimental support. Segre and Kaeberie (12) found that both specific antibody and "normal" gamma globulin enhance the antibody response of newborn piglets deprived of colostmm. I-Iemphill et al. (13) terminated tolerance to human serum albumin in mice by injecting antigen-antibody complexes. M/iller and Wigzell (7) found a minor enhancement of the number of plaque-forming cells in occasional mice when small amounts of specific antibody were administered prior to an injection of sheep red cells. Williams (14) has demonstrated that the reduced follicular localization of polymerized flagellin observed in X-irradiated and lymphocyte-depleted rats was significantly improved when specific antisera were injected prior to antigen injection. In these studies, the immunoglobulin type of the enhancing antibody was not determined. More recently, Pearlman (15) has reported that whereas both 19S and 7S antibodies are effective inhibitors of antibody formation, both are also capable of enhancement when given in small amounts. In contrast to the depressive effect, enhancement appeared to be nonspecific. Others have reported that, though small amounts of IgG anti-Rh antibody can suppress the response of Rh-negative mothers to the Rh-positive ceils of their newborn, relatively small amounts of IgM antibody actually result in enhanced antibody formation (16).The present studies were initiated in the hope of clarifying some of the questions raised by our studies of the primary immune response of mice to 133

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In these experiments, we show that the interaction of antigen and B cell surface immunoglobulin is not essential for the generation of an IgG in vitro response to the hapten p-azophenyl-lactoside (lac). In our experimental system, keyhole limpet hemocyanin (KLH) was first selectively attached either to H-2, Ia or Ig receptors of lac-primed B cells by a hapten sandwich technique or to Fc receptors by complexes of azophenyl arsonate (ars)-coupled KLH and anti-ars. The labeled cells were then cultured with KLH-specific T cells for 5 days in the absence of antigen. Under all conditions of attachment we observed a significant anti-lac IgG response. We have demonstrated an absolute requirement for KLH-specific helper T cells. The results thus indicate that T helper cells are by themselves, regardless of the B cell antigen that serves to effect bridging, sufficient to activate B memory cells. We could find no evidence to support either a matrix theory or a two-signal hypothesis as currently proposed.

The immune capabilities of the Peyer's patches have been investigated by the use of an in vitro system. Despite our failure to stimulate Peyer's patch lymphocytes in vivo it appears that Peyer's patches behave immunologically as peripheral lymphoid tissues. Cultures prepared from the dissociated Peyer's patches of normal rabbits respond to sheep erythrocytes. The response is comparable to that obtained with spleen cultures from the same animals and is not dependent on the presence of the epithelial cells which line the lumen. Similar thymic cultures do not respond. Our experiments with cultures prepared from rabbits which have received one or two injections of SRC show that the Peyer's patches contain both IgM and IgG "memory" cells which have migrated from the spleen. The concentration of these cells in the spleen remains several hundredfold higher.

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...