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The secondary immune response to a hapten in vitro. Antigen concentration and the carrier effect.

The in vitro secondary stimulation of the production of anti-hapten antibody has been analyzed with a view to elucidating the role of the carrier molecule and cell-to-cell interactions in this response. Stimulation was carried out on fragment cultures of the spleens of irradiated BALB/c mice which had been reconstituted with 3-4 x 10(7) spleen cells from isologous mice previously immunized with DNP-Hy. The results indicated that the response was maximized by stimulation with DNP-Hy, the homologous complex, however anti-DNP antibody production could be obtained by stimulation with DNP on several nonhomologous carriers including poly-D-lysine, a poor immunogen. The results also indicated that while DNP-Hy and DNP-nonhomologous-carrier complexes were stimulatory at equally low DNP concentrations, at DNP concentrations over 10(-6)M DNP-Hy was stimulatory, while DNP on nonhomologous carriers was inhibitory. The results are interpreted as indicating that: (a) the affinity of the antigen-cell interaction is more likely determined by multivalent binding than by carrier recognition, (b) that a stimulatory interaction of a polyvalent antigen with a B-lymphocyte cannot be excluded, (c) that if cell-to-cell interaction is necessary for stimulation, then both cells may recognize the same determinant, and (d) that the marked enhancement of antigenic stimulation attributable to carrier recognition may result from stimulatory interactions of cells recognizing different antigenic determinants. A mechanism is postulated whereby stimulation is dependent on the formation of stable complexes resulting from two cells sharing in the binding of numerous antigen molecules. Cells recognizing carrier determinants would increase the probability of such interactions particularly at high antigen concentrations.

Radloimmunoassay.--The radioimmunoassay used to measure antibody concentrations and the purification of the mouse anti-DNP antibody used to standardize this assay have been previously described (13). 30/zg of DNP-Iys-BAC was used as the immunoadsorbent for each analysis and 125I-rabbit anti-mouse immunoglobulin Fab fragment (13) was used to detect bound anti-DNP antibody. 0.1 ml of culture fluid was used for each antibody analysis. The radioimmunoassay for anti-Hy antibody was also carried out as previously described (14).
Mouse Immunlzatlons.--BALB/c mice 2-3 months of age were immunized by the intraperitoneal injection of 0.1 mg of I)NP-Hy in complete Freund's adjuvant. Sera antibody concentrations, assayed by the radioimmunoassay, showed a maximum concentration of 0.3 mg/ml 2-4 wk after immunization. Sera anti-DNP antibody concentrations were less than 20 ng/ml 4-8 months after immunization when the animals were used as spleen cell donors or were secondarily immunized in vivo. Secondary immunizations were carried out by intraperitoneal injections with 50 #g of DNP-Hy in PBS.
Cell Transfers.--Spleens were removed and cells were separated by a teflon pestle tissue homogenizer. Cells were washed and resuspended in Dulbecco's modified Eagle's medium (Grand Island Biological Co., Grand Island, N. Y.). 30-40 million cells in 0.3 ml doses were injected into a tail vein of syngenic recipients irradiated with 900 R 24 hr previously. Nonimmunized controls were obtained by injecting 4 )< l0 T cells obtained from spleens of nonimmunized mice.
Fragment Cultures.--24 hr after cell transfer spleens were removed and cut into 1-1.2 mm cubes. Culture conditions were similar to those previously described (14,15). Each fragment was placed in 0.5 ml of Dulbecco's modified Eagle's medium supplemented with 50 #1 of horse serum from which gamma globulin was removed (Hyland Laboratories, Yonkers, N. Y.), 25/zl of 10 day chick embryo extract (a gift from Dr. Jay Lash), 50 units of penicillin, and 50 #g of streptomycin. Cultures were incubated in a moist chamber at 37°C in an atmosphere of 93% 02 and 7% CO2 . Media were removed and replaced at 2-3 day intervals for the duration of the cultures. Antigen was added to the culture fluid 24 hr after cultures were initiated. Fragments were rinsed free of antigen containing culture fluid 2-4 days after antigen was added. For inhibition studies, the inhibitor was added to the fragment cultures at least 1 hr before antigen was added and was removed at the time of antigen removal.
Collected culture fluids were analyzed for anti-DNP and anti-Hy antibody content by the radioimmunoassay. Results are presented as the average amount of antibody produced per fragment per day. Included in this average were all fragments stimulated under the stated conditions in at least three separate experiments. In each experiment 4 fragments were utilized for each experimental point so that the data presented represented the results of at least 12 fragments for each point. In general a single experiment consisted of 100-120 fragments obtained from four to six recipient mice which had received pooled spleen cells from two donor mice.
Equilibrium Dialysis.--Equilibrium dialysis was carried out as previously described (12,13). Sera and culture fluids were twice precipitated with 40% saturated ammonium sulfate and dialyzed against PBS before analysis. Fig. 1 shows the amount of anfi-DNP antibody released by fragments at various times after stimulation with 0.1 mg of DNP-Hy. Antibody was first detected at day 6-8 and reached a maximum at day 12-14. Such a response was observed in at least 80% of fragments tested. Antibody production continued for at least a month in these cultures. If no DNP-containing antigen was added to culture fluids or if cells from nonimmunized mice were used as donor cells, no anti-DNP antibody production was observed. Similar results were obtained when culture fluids from these fragments were analyzed for anti-Hy production. At least 80% of the fragments produced anti-Hy antibody with an average maximum release of 70 ng/day at day 14.

RESULTS
Stimulation by DNP-Hy was partially inhibited when carried out in the presence of relatively high concentrations of DNP-lysine or Hy. Table I summarizes the data obtained from cultures stimulated in the presence of free hapten or carrier. These results are consistent with those previously reported by several investigators (16)(17). 2 Control studies indicated that 10-SM DNPlysine did not reduce the amount of anti-Hy antibody produced by cultures stimulated with DNP-Hy. Fig. 2 shows the effect on stimulation of variations in the carrier used in the stimulatory complex and the concentration of the various antigens used. Fig. 3 presents the same data calculated on the basis of DNP molarity. Maximum stimulation was obtained with 0.1 tzg/ml of DNP-Hy; however, significant stimulation was obtained with concentrations as high as 100 #g/ml and as low as 10 -6 #g/ml. Significant stimulation was also observed with DNP-HSA and DNP-B~G at concentrations as high as 1/~g/ml and as low as 10 -G b*g/ml. DNPpoly-L-lysine stimulated at concentrations as high as 0.03/~g/ml and as low as homologous carriers including DNP-poly-I)-lysine, a poor immunogen, (c) stimulation was obtained with the hapten on homologous and nonhomologous carriers at antigen concentrations as low as 10 -8 #g/ml (10-last DNP), and (d) at an antigen concentration of 10/~g/ml (10-6M DNP) and greater, DNP-Hy was stimulatory while DNP on nonhomologous carriers specifically inhibited the response to DNP. Several investigators have reported the in vivo stimulation of anti-hapten antibody by hapten on nonhomologous carriers (18,19). However, other investigators have found that hapten on the homologous carrier is a much better immunogen and have implied that specific recognition of carrier determinants enhances the affinity of hapten recognition (20).
The demonstration, in this report, that stimulation of a secondary response was obtained at equally low concentrations of the hapten on homologous and nonhomologous carriers indicates that the affinity of the hapten cell interaction was independent of the carrier molecule. The affinity of the antibody produced after stimulation with DNP-Hy in a concentration of 10 -~/~g/ml (10-1° M DNP) was 2.6 X 107 ~r -1. Since the hapten was presented in a multivalent form, it is possible that the affinity of cell-bound antibody for hapten was greatly enhanced by multivalent binding (21)(22)(23)(24). Thus cell-bound antibody of an affinity of 2.6 X 107 M -1 for a monovalent hapten could bind a multivalent antigen with an affinity more than two logarithms higher. From the results presented here such multivalent interactions appear more likely to explain the stimulatory capacity of low antigen concentrations than would specific interactions with carrier determinants.
On the other hand, specific recognition of the homologous carrier appeared highly significant at higher antigen concentrations. DNP on nonhomologous carriers specifically inhibited the response to DNP at concentrations where DNP-Hy was stimulatory. Thus while a stimulatory interaction of the haptenic determinant in a polyvalent form and an immunocompetent cell may be possible, the general mechanism of antigenic stimulation is probably more complex.
Mitchison and his co-workers consider the carrier effect as a function of thymus-dependent cells (T-lymphocytes) which recognize carrier determinants. Such cells presumably bind the hapten-carrier complex and present the hapten in a concentrated form to bone marrow-derived cells (B-lymphocytes) which recognize the hapten and upon stimulation produce antibody against it (3). Two cells which recognize determinants on the same antigen molecule could possibly form a highly stable complex if both shared in the simultaneous binding of numerous antigen molecules (21)(22)(23)(24). Cell-to-cell interactions could thus play a role in antigenic stimulation if such stable complexes between antigen and cells were necessary. Such interactions would be minimized at very low antigen concentrations where too few antigen molecules would be available for stable cross-linking and at very high antigen concentrations where cross-linking would be minimized by blockage of antigen binding sites. Stimulation of anti-DNP antibody production, in vitro, by low concentrations of hapten on homologous and nonhomologous carriers could result from interactions of two cells recognizing DNP. Since this determinant is polyvalent it may be capable of simultaneous interaction with cell-bound antibody of more than one cell. Because of its polyvalence, its interaction with cell-bound antibody may be of relatively high affinity. Thus saturation of DNP receptor sites may be reached at relatively low antigen concentrations. Receptor sites recognizing carrier determinants, however, may still be available for cross-linkage at higher antigen concentrations. Thus the recognition of Hy would enhance stimulation of anti-DNP cells at high antigen concentrations. This is consistent with the finding that high concentrations of Hy-inhibited stimulation with DNP-Hy.
This mechanism for stimulation through cell-to-cell interaction does not differentiate between the types of cells involved. It is possible that the interaction of two B-lymphocytes could be mutually stimulatory. Such a mechanism has been postulated for the stimulation of "mono-focal" antibody production where stimulation shows enhancement by carrier recognition but can occur in the absence of carrier specific cells (14). On the other hand, the presence of T-lymphocytes recognizing determinants on the antigen molecule could serve to increase the probability of productive interactions of antigen and hapten specific B-12clnphocytes. This would be consistent with the marked enhancement of antigenic stimulation generally observed in the presence of carrier recognition. An interesting correlate of the above hypothesis is the possibility that tolerance may be produced by simple antigen-cell interactions or by the formation of unstable complexes between two cells cross-linked by only a few antigen molecules. Such a state would be obtained at very high or very low antigen concentrations as has been described for the induction of the tolerant state (25). SUMMARY The in vitro secondary stimulation of the production of anti-hapten antibody has been analyzed with a view to elucidating the role of the carrier molecule and cell-to-cell interactions in this response. Stimulation was carried out on fragment cultures of the spleens of irradiated BALB/c mice which had been reconstituted with 3-4 X l0 T spleen cells from isologous mice previously immunized with DNP-Hy. The results indicated that the response was maximized by stimulation with DNP-Hy, the homologous complex, however anti-DNP antibody production could be obtained by stimulation with DNP on several nonhomologous carriers including poly-B-lysine, a poor immunogen. The results also indicated that while DNP-Hy and DNP-nonhomologous-carrier complexes were stimulatory at equally low DNP concentrations, at DNP concentrations over 10 -6 M DNP-Hy was stimulatory, while DNP on non-homologous carriers was inhibitory. The results are interpreted as indicating that: (a) the affinity of the antigen-cell interaction is more likely determined by multivalent binding than by carrier recognition, (b) that a stimulatory interaction of a polyvalent antigen with a B-lymphocyte cannot be excluded, (c) that if cell-to-cell interaction is necessary for stimulation, then both cells may recognize the same determinant, and (d) that the marked enhancement of antigenie stimulation attributable to carrier recognition may result from stimulatory interactions of cells recognizing different antigenic determinants. A mechanism is postulated whereby stimulation is dependent on the formation of stable complexes resulting from two cells sharing in the binding of numerous antigen molecules. Cells recognizing carrier determinants would increase the probability of such interactions particularly at high antigen concentrations.