VARIATION IN ACCESSIBLE CELL SURFACE IMMUNOGLOBULIN AMONG ANTIBODY-FORMING CELLS

Spleen cells from CBA mice that had been primarily or secondarily immunized with sheep red blood cells were reacted at 0°C with a 125I-labeled polyvalent rabbit anti-mouse globulin reagent. After suitable washing, the cells were placed in a plaque-revealing monolayer and warmed to 37°C. Plaques appeared within 10–20 min. Single plaque-forming cells (PFC) were taken from the middle of plaques, were washed by micromanipulation, and were singly dried on glass slides. The amount of attached antireceptor was assessed by quantitative radioautography. Great variation in "receptor density" was encountered among the 258 single cells studied. However, early, immature PFC in both primary and secondary responses had statistically significantly more receptors than late, mature PFC. On any given day point, no difference was found between IgM- and IgG-forming cells. The results were consistent with the view that cells still able to be driven to further proliferation by antigen retain receptors, and conversely that cells, as they mature, lose both receptors and ability to be influenced by antigen

EFFECTOR CELL BLOCKADE : A NEW MECHANISM OF IMMUNE HYPOREACTIVITY INDUCED BY MULTIVALENT ANTIGENS

This study describes the effects of incubating antibody-forming cells (AFC), either as mass cell suspensions, or as single AFC in microdroplets, with antigens against which the cells display specificity. Most of the work was done with hapten-specific anti-DNP-AFC, but AFC with specificity against flagellar antigens or fowl gamma globulin (FGG) were also included. It was noted that 30-min incubation of AFC with highly multivalent forms of antigen caused a substantial partial suppression of the antibody-forming performance of the AFC as measured by a hemolytic plaque test. Thus, when cell suspensions containing anti-DNP plaque-forming cells (PFC), were incubated for 30 min at 37°C with 100 µg of DNP-polymerized flagellin (DNP-POL), the number of plaques appearing after washing of the cells and placing them in plaque-revealing erythrocyte monolayers was reduced to 50% or less compared with the number of plaques observed with control portions preincubated with medium alone. Preincubation with DNP-lysine, with oligovalent DNP-protein conjugates, or with irrelevant antigens produced no such inhibition. Studies where preinhibited PFC suspensions were mixed with control suspensions before assay showed that a nonspecific carryover of antigen into the assay system was not involved. The inhibitory effect could also be initiated by holding cells at 0°C with DNP-POL, but in that case, inhibition only became manifest after cells were incubated for 30 min at 37°C before being placed in plaque-revealing monolayers. This suggested that inhibition was initiated by adsorption of multivalent antigen onto PFC-surface Ig, but required some active process before secretion actually slowed down. The effect was dose- and time-dependent, antigen-specific, and generalized for all antigens studied. As well as yielding reduced plaque numbers, the preinhibited cells also gave smaller, more turbid plaques, suggesting a reduction in antibody-forming rate by each PFC rather than the elimination of PFC. Consistent with this suggestion was the observation that the degree of inhibition of plaque formation could be increased by decreasing the sensitivity of the assay so that only AFC secreting at high rates were detected. A micromanipulation study, where single PFC were subjected to inhibition, and were then tested for the rate at which they could cause hemolysis, showed a 68% inhibition of mean secretory rate. Micromanipulation studies were performed to test the amount of cell surface-associated Ig on control and preinhibited PFC. For this, single PFC were held with [125I]antiglobulin and quantitative radioautography was performed. No significant difference emerged, suggesting that retention of secreted Ig on cell-attached antigen was not the cause of inhibition. The results are discussed in the framework of tolerance models and blocking effects at the T-cell level by antigen-antibody complexes. The name effector cell blockade is suggested in the belief that the phenomenon may be a general one applying to both T and B cells

QUANTITATIVE FEATURES OF A SANDWICH RADIOIMMUNOLABELING TECHNIQUE FOR LYMPHOCYTE SURFACE RECEPTORS

The present study was designed to devise and characterize an indirect or sandwich radioimmunolabeling technique for the study of lymphocyte surface receptors of immunoglobulin nature. Mouse lymphocytes from various sources were treated by the method of Shortman et al. to remove debris and damaged cells. This was an important preliminary step, as without it, little meaning could be attached to bulk scintillation counting of labeled cell suspensions, in view of the marked tendency of dead or damaged cells to adsorb protein nonspecifically. Next, cells were reacted at 0°C for 30 min with graded dilutions of unlabeled rabbit antisera against defined mouse Ig chains. After washing, the cells were reacted with a sheep anti-rabbit globulin reagent labeled with 125I, again at graded concentrations. After further washing, lymphocyte labeling was quantitated by both bulk scintillation counting and radioautography. Conditions were defined in which nonthymus-derived cells (B cells) but not thymus-derived cells (T cells) could be labeled. Most B cells displayed κ- and µ-chains on their surface, but some also displayed α- and γ2-chains, though in smaller amounts. When the concentration of both the first and the second reagents were raised considerably, conditions were defined under which virtually all T cells could be labeled by polyvalent antiglobulin sera, anti-κ sera, or, with more difficulty, by anti-µ sera. A large series of control experiments confirmed the serologic specificity of this labeling. It was shown that under equivalent conditions, B cells bind 100–400 times more antiglobulin than do T cells. The theoretical implications of the results are briefly discussed. It is argued that the sandwich approach offers certain technical advantages over direct labeling procedures for further analyses of T cell receptors and for studies of receptor metabolism

INDUCTION OF B CELL TOLERANCE IN VITRO TO 2,4-DINITROPHENYL COUPLED TO A COPOLYMER OF D-GLUTAMIC ACID AND D-LYSINE (DNP-D-GL)

Spleen cells from CBA or congenitally athymic ("nude") mice were pretreated with various concentrations of DNP coupled to a copolymer of D-glutamic acid and D-lysine (DNP37-D-GL), under various conditions of time and temperature. After washing, they were then cultured for 3 days with the direct B cell immunogen, DNP coupled to Salmonella adelaide flagella (DNP-FLA). Under all circumstances tried, exposure of cells to 1 µg/ml DNP-D-GL caused a 70–100% depression in the subsequent DNP-specific PFC response, and 100 ng/ml caused a lesser but still substantial effect. At the concentrations used, DNP-D-GL did not affect irrelevant antibody responses. Though cells from nude mice responded somewhat less well to DNP-FLA than those from CBA mice, no significant difference in the reaction of the two populations to the tolerogen was noted. This demonstrates that DNP-D-GL can, as previously suspected, directly cause unresponsiveness in B lymphocytes