CELL INTERACTIONS IN THE IMMUNE RESPONSE IN VITRO : II. THE REQUIREMENT FOR MACROPHAGES IN LYMPHOID CELL COLLABORATION

The requirement for macrophages in thymus-dependent antibody responses was studied in vitro. Three different macrophage-deficient cell populations were studied: spleen cells passed through a glass bead column at 37°C, spleen cells cultured with specific antimacrophage serum, and thoracic duct lymphocytes. These cell populations from mice primed to dinitrophenylated (DNP) fowl gamma globulin were unable to respond to the homologous conjugate in vitro. DNP-reactive B cells were present in normal proportions, since all three macrophage-depleted populations responded normally to macrophage-independent and thymus-independent DNP flagella. Carrier-reactive T cells were present, as the helper capacity of carrier-primed spleen cells was the same as carrier-primed lymphocytes, and thoracic duct lymphocytes are a well-established source of helper cells. The inhibition of the cooperative response was thus due to removal of macrophages, and this was proven by restoration of thymus-dependent anti-DNP responses by small numbers of anti-θ-treated peritoneal exudate cells. These results suggest that macrophages are essential in cell collaboration, While their exact function in cell collaboration is not yet known, the above observation suggests that the mechanism of T-B collaboration involves the surface of macrophages

INDUCTION OF IMMUNITY AND TOLERANCE IN VITRO BY HAPTEN PROTEIN CONJUGATES : III. HAPTEN INHIBITION STUDIES OF ANTIGEN BINDING TO B CELLS IN IMMUNITY AND TOLERANCE

The capacity of dissociated spleen cell suspensions to be immunized by dinitrophenylated polymeric flagellin (DNP POL), in the absence of thymus-dependent lymphocytes or macrophages, provided a simple experimental system to investigate the mechanism of binding of antigen molecules to nonthymus-dependent lymphocyte (B cell) receptors during the induction of immunity or tolerance. Various nonimmunogenic DNP compounds were used to inhibit the anti-DNP response to DNP POL. By performing inhibition experiments of brief duration at 4°C, it was established that the inhibition of the anti-DNP response by nonimmunogenic compounds was due to competitive blockade of receptors, and not tolerance or receptor modulation. It was found that univalent DNP compounds were much less efficient inhibitors of the antibody response than multivalent DNP conjugates. The difference in inhibitory capacity between univalent and multivalent DNP human globulin (DNP HgG) suggested the importance of interaction with both combining sites of a single receptor antibody molecule. Nonimmunogenic highly conjugated DNP3POL was a more efficient inhibitor of the anti-DNP response to immunogenic DNP1POL than DNP12HgG, indicating that interactions at more than one receptor molecule are involved in immunization of B cells. Recent demonstrations of the rapid metabolic turnover of receptor antibody molecules suggests that the requirement for multipoint binding (to different receptors) may simply be to maintain the antigen at the cell surface in a dynamic system. Competitive inhibition experiments were also performed to investigate the mechanism of binding of DNP3POL in the induction of B cell tolerance. It was found that monovalent DNP compounds or multivalent DNP12HgG did not prevent the induction of tolerance, unlike their capacity to inhibit immunity, suggesting that a tolerance-inducing antigen binds more avidly to the cell membrane than an immunogen. The inhibition data obtained here, together with prior results describing the differential immunogenicity of DNP conjugates of different structure, and the importance of epitope density on DNP POL conjugates, permit certain conclusions about the details of antigen-receptor interaction in immunity and tolerance. Distinctions between the mechanisms of immune and tolerance induction are discussed

CELL INTERACTIONS IN THE IMMUNE RESPONSE IN VITRO : V. SPECIFIC COLLABORATION VIA COMPLEXES OF ANTIGEN AND THYMUS-DERIVED CELL IMMUNOGLOBULIN

The mechanism of interaction of T and B lymphocytes was investigated in an in vitro hapten carrier system using culture chambers with two compartments separated by a cell impermeable nucleopore membrane. Because specific cell interaction occurred efficiently across this membrane, contact of T and B lymphocytes was not essential for cooperation which must have been mediated by a subcellular component or "factor." By using different lymphoid cell populations in the lower culture chamber and activated thymus cells in the upper chamber (with antigen present in both), it was found that the antigen-specific mediator acted indirectly on B cells, through the agency of macrophages. Macrophages which had been cultured in the presence of activated T cells and antigen acquired the capacity to specifically induce antibody responses in B cell-containing lymphoid populations. Trypsinization of these macrophages inhibited their capacity to induce immune responses, indicating that the mediator of cell cooperation is membrane bound. By using antisera to both the haptenic and carrier determinants of the antigen as blocking reagents, it was demonstrated that the whole antigen molecule was present on the surface of macrophages which had been exposed to activated T cells and antigen. Because specifically activated T cells were essential a component of the antigen-specific mediator must be derived from these cells. By using anti-immunoglobulin sera as inhibitors of the binding of the mediator to macrophages, the T cell component was indeed found to contain both κ- and µ-chains and was thus presumably a T cell-derived immunoglobulin. It was proposed that cell cooperation is mediated by complexes of T cell IgM and antigen, bound to the surface of macrophage-like cells, forming a lattice of appropriately spaced antigenic determinants. B cells become immunized by interacting with this surface. With this mechanism of cell cooperation, the actual pattern of antigen-B cell receptor interactions in immunization would be the same with both thymus-dependent and independent antigens. An essential feature of the proposed mechanism of cell cooperation is that macrophage-B cell interaction must occur at an early stage of the antibody response, a concept which is supported by many lines of evidence. Furthermore this mechanism of cell interaction can be elaborated to explain certain phenomena such as the highly immunogenic macrophage-bound antigen, antigenic competition, the distinction between immunity and tolerance in B lymphocytes, and the possible mediation of tolerance by T lymphocytes

INDUCTION OF IMMUNITY AND TOLERANCE IN VITRO BY HAPTEN PROTEIN CONJUGATES : I. THE RELATIONSHIP BETWEEN THE DEGREE OF HAPTEN CONJUGATION AND THE IMMUNOGENICITY OF DINITROPHENYLATED POLYMERIZED FLAGELLIN

Of many dinitrophenylated (DNP) protein conjugates tested, only DNP conjugated to polymerized flagellin (DNP-POL) (or the structurally related bacterial flagella) elicited a primary anti-DNP response in vitro. Other DNP proteins, such as DNP-monomeric flagellin (DNP-MON), were capable of inducing secondary responses in vitro. The capacity of DNP-POL to immunize spleen cell suspensions devoid of thymus-derived cells was the reason for the greater immunogenicity of DNP-POL, since even large numbers of flagellin-reactive activated thymus cells did not increase the anti-DNP response of normal spleen cells immunized with DNP-POL, whereas the thymus-dependent response to DNP-MON was markedly increased. The capacity of various batches of DNP-POL to immunize normal spleen cells in vitro varied markedly, depending on the number of DNP groups conjugated. DNP-POL with few DNP groups conjugated was immunogenic, but even at very high concentrations did not induce tolerance. In contrast, highly conjugated DNP-POL did not immunize, but readily induced tolerance. DNP-POL with intermediate degrees of conjugation were, like unconjugated polymeric flagellin, capable of inducing both immunity and tolerance. Since DNP-POL immunizes bone marrow-derived lymphocytes (B cells) directly the reduced response was not due to a masking of carrier determinants, necessary for cell collaboration. By using mixed DNP-5-(dimethylamino)-1-naphthalyl (dansyl)-POL conjugates, it was found that the inhibitory effect of a high degree of hapten conjugated was hapten specific. Depolymerization of DNP-POL to DNP-MON, which does not induce primary anti-DNP responses, was excluded by centrifugation analysis and electron microscopy. The relationship of the degree of hapten conjugation on DNP-POL to the capacity to induce tolerance and immunity in B cells has clarified the mechanism of immunological triggering of these cells. A model of the mechanism of "signal" discrimination between immunity and tolerance in B cells, based on these findings, is proposed

ANTIBODY-MEDIATED SUPPRESSION OF THE IMMUNE RESPONSE IN VITRO : I. EVIDENCE FOR A CENTRAL EFFECT

Antibody-mediated suppression of the in vitro immune response to polymerized flagellin of Salmonella adelaide and to sheep erythrocytes was studied at the cellular level. Normal mouse spleen cells, preincubated in vitro with mixtures of antigen and antibody for short periods of time before being washed, did not respond to an optimal antigenic challenge in vitro, whereas similar cells treated with antibody alone gave a normal response. The degree of immune suppression was found to depend on the time of preincubation. Significant immune suppression could be induced in as short a time as 15 min, whereas profound suppression (90%) required the incubation of cells with mixtures of antigen and antibody for 4–6 hr. Mouse spleen cells treated similarly were also unable to respond subsequently to the antigen upon transfer to lethally irradiated hosts, as measured at both the level of the antigen-reactive cell and that of serum antibody production. These results were taken as evidence that in vitro an effect of antibody-mediated suppression occurred at the level of the immunocompetent cell. Similarities between immune tolerance and antibody-mediated suppression in vitro were described, and the significance of the findings discussed in the light of current concepts of the mechanism of antibody-mediated suppression

ANTIBODY-MEDIATED SUPPRESSION OF THE IMMUNE RESPONSE IN VITRO : II. A NEW APPROACH TO THE PHENOMENON OF IMMUNOLOGICAL TOLERANCE

Immunological tolerance to H antigens of Salmonella adelaide may be induced in vitro by the exposure of mouse spleen cells for 6 hr to an immunogenic dose of polymerized flagellin in the presence of low concentrations of specific antibody. Such antibody-mediated tolerance requires an optimal antigen: antibody ratio for its induction. A shift in this ratio in favor of the antibody concentration results in failure of tolerance induction and leads to immune suppression commonly known as antibody-mediated feedback inhibition which is not analogous to immunological tolerance. Fragment A of flagellin fails to induce immunological tolerance in vitro. Tolerance to polymerized flagellin may however be induced in vitro, provided the spleen cells are exposed to fragment A in the presence of specific antibody for 6 hr. The results are discussed in the light of current theories of the mechanism of tolerance induction

THE RELATIONSHIP BETWEEN ANTIGENIC STRUCTURE AND THE REQUIREMENT FOR THYMUS-DERIVED CELLS IN THE IMMUNE RESPONSE

Certain antigens such as polymerized flagellin are capable of producing relatively normal antibody levels in thymectomized mice, whereas others, including heterologous erythrocytes require the presence of T cells in a helper capacity. The mechanism of thymus-independent antibody production was investigated by comparing the primary IgM responses of spleen cells from ATXBM, XBM, and normal mice to various physical forms of the flagellar antigens of Salmonella adelaide in vitro. No reduction in antibody-forming cell levels to polymerized flagellin over a wide dose range was observed in ATXBM cultures, although the same spleen cells did not respond to an optimal dose of sheep red cells. In contrast, when flagellar determinants were presented in a monomeric form or as flagellin-coated donkey red cells, a highly significant difference was observed between the antibody responses of spleen cells from ATXBM mice and XBM or normal controls. The results suggested that the requirement for T cells in antibody production is not a property of specific antigenic determinants, but depends on the mode of antigenic presentation. The validity of this conclusion was confirmed by using another antigenic determinant (DNP) coupled either to the thymus-independent carrier, POL, or to the thymus-dependent carrier, DRC. Spleen cells from XBM mice produced comparable AFC levels to both forms of DNP, but the results from ATXBM cultures showed a marked difference. The anti-DNP response to DNP-DRC was greatly reduced compared to controls, whereas that to DNP-POL was normal even after prolonged thoracic duct drainage of the ATXBM donors and pretreatment of their spleen cells with anti-θ-serum and complement. The data presented here imply that the role of T cells in humoral immunity is the presentation of antigen to B cells in such a manner as to initiate optimal antibody synthesis