Antibody production in mice: II. The mechanism of antigenic stimulation in the secondary immune response

To study the mechanism of antigenic stimulation in the secondary immune response, primed lymphoid cells were stimulated with antigen in various ways in vitro. The effect of antigenic stimulation was assessed by the antibody titres obtained after in vivo culture of primed cells in X-irradiated recipients.

Primed cells were much more effectively stimulated by antigen—antibody (Ag—Ab) complex than by free antigen. Primed cells could also be stimulated by spleen or lymph node cells from normal mice which had been exposed to free antigen or Ag—Ab complex in vitro or in vivo and thoroughly washed. Under these conditions, Ag—Ab complex was again much more effective than free antigen. When the cells were incubated with Ag—Ab complex, the dose of antigen bound to the cells was somewhat increased. But this increased binding of antigen could not solely account for the increase in immunogenicity.

It is suggested that the ingestion of antigen by macrophages is facilitated by the presence of antibody and that the macrophages mediate the effective immune stimulus to memory cells.

The effect of antibody in increasing the immunogenicity of antigen was lost completely when antibody was digested with pepsin. Thus, the Fc portion of antibody seemed to be important for this effect. However, it was demonstrated that antibody does not operate by becoming attached to macrophages as cytophilic antibody, and that complement is not involved in this process. The augmenting mechanism of antibody on the antigenic stimulation mediated by macrophages was discussed.

     

Antibody production in mice: VI. Effect of anti-carrier antibody on cellular co-operation in the primary anti-hapten antibody response

We studied the effect of passively administered anti-carrier and anti-hapten antibodies on the primary anti-hapten antibody response to hapten—carrier conjugates in mice. Bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole limpet haemocyanin (KLH) were used as carrier molecules, and 2,4-dinitrophenyl (DNP) group was used as a haptenic determinant.

Three groups of mice were injected intravenously with anti-carrier antiserum, anti-hapten antiserum and normal serum as the control, respectively, immediately after the immunization with the hapten—carrier conjugate.

The primary anti-carrier antibody response was markedly suppressed by the passively administered anti-carrier antibody but not anti-hapten antibody. However, the primary anti-hapten antibody response was suppressed not only by passively administered anti-hapten antibody but also by the injection of anti-carrier antibody in an early period after the immunization.

When anti-carrier antibody was given twice 0 and 7 days after immunization, the primary anti-hapten antibody response was markedly suppressed and the suppressive effect was observed even at a later period. In contrast, anti-hapten antibody given by the same schedule as above suppressed only the primary anti-hapten antibody response, but not the anti-carrier antibody response.

Passively administered anti-carrier antibody did not suppress carrier-specific helper cell development, but still suppressed the development of B memory cells and antibody formation against carrier determinants. The antigen dose required for the development of B-cell memory was much higher than that necessary for the stimulation of T cells.

Passively administered anti-carrier antibody clearly inhibited the cellular cooperation between carrier-committed helper cells and hapten-specific B cells and the augmented primary anti-hapten antibody response induced by carrier-primed T cells was clearly abolished. Furthermore, mice preimmunized with carrier showed significantly lower anti-hapten antibody response following the hapten—carrier challenge. Moreover, development of hapten-specific memory cells was also suppressed.

Thus, even in the non-specific antibody-induced suppression by anti-carrier antibody, negative feedback effect of a B-cell product with anti-carrier specificity exclusively regulates the B-cell line development and differentiation.

     

Antibody production in mice: IV. The suppressive effect of anti-hapten and anti-carrier antibodies on the recognition of hapten-carrier conjugate in the secondary response

Non-cross reactive antigens, bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole-limpet haemocyanin (KLH), were used as the carrier proteins conjugated with 2,4-dinitrophenyl (DNP) and benzylpenicilloyl (BPO) groups as haptenic determinants. Lymphoid cells obtained from mice primed with these hapten—protein conjugates were stimulated in vitro with various DNP- and BPO-proteins as the second antigen and transferred into X-irradiated recipients.

A good secondary antihapten antibody response was obtained when the cells primed with a hapten-carrier conjugate were stimulated with the same conjugate as that used for priming, but no response could be obtained if carrier for the secondary stimulus differed from that used for priming. However, stimulation with hapten-heterologous carrier gave a good secondary response to the hapten provided that cells committed to the heterologous carrier were also present. The activity of carrier-primed cells could not be replaced by circulating antibody. Co-operation between the carrier-primed cells and the hapten-primed cells was also observed when the antigenic stimulus was hapten-carrier conjugate incorporated into macrophages. The immunogenic molecule from the macrophage was considered to consist of the haptenic group and at least a portion of the carrier.

When the primed cells were secondarily stimulated with hapten-carrier conjugates incorporated into macrophages and the mixtures transferred to recipient mice, the passive administration of antihapten antibody to these recipients specifically suppressed the corresponding antihapten antibody response only but passively administered anticarrier antibody completely suppressed both the anti-carrier and antihapten antibody responses. The suppression of antihapten antibody response by anticarrier antibody seems to be the result of failure of co-operation between carrier-primed cells and hapten-primed cells.

     

Regulation of anti-hapten antibody response by chemically modified carrier antigen preferentially provoking delayed-type hypersensitivity: I. Possible T—T cell interaction in the suppression of antibody response

The i.p. immunization with chemically modified antigen (dodecanoyl-bovine serum albumin, d-BSA) emulsified in Freund's incomplete adjuvant (FIA) of CBA mice provoked delayed-type hypersensitivity (DTH), but not any detectable formation of antibody to the original antigen (BSA). Furthermore, it was found that immunization with d-BSA could generate T cells capable of inhibiting the antibody response to hapten on BSA, and the immunosuppressive effects of these T cells were presumably not due to direct action on hapten-primed and antibody producing B cells. These results were obtained from the following experiments: (1) anti-hapten antibody response to dinitrophenylated-BSA (DNP-BSA) was inhibited when the mice had been primed previously with d-BSA in FIA. This inhibition was regulated by the specificity of the carrier, since the mice treated with d-BSA did not inhibit the anti-DNP antibody response after the immunization with DNP-heterologous carrier, i.e. DNP-keyhole limpet haemocyanin (DNP-KLH). (2) The passive transfer of spleen cells, which had been obtained from donors primed with d-BSA in FIA, inhibited the primary anti-DNP antibody response of syngeneic mice after immunization with DNP BSA. (3) Injection of d-BSA-primed spleen cells suppressed an adoptive anti-DNP antibody response in mice which had been irradiated and had previously had their immunocompetence reconstituted by the cell transfers with both DNP-primed and BSA-primed spleen cells. This in vivo immunosuppressive effect of d-BSA-primed spleen cells did not act on hapten-primed B cells, since d-BSA-primed spleen cells could not suppress the adoptive secondary antibody response reconstituted by DNP-primed cells and bacterial alpha-amylase (BαA)-primed cells. This finding suggests that a T—T cell interaction exists for the suppression of the anti-DNP antibody response to DNP-BSA by d-BSA-primed cells.     

Differences in interaction between immune complexes and complement receptors in serum and cerebrospinal fluid

Antigen—antibody complexes (Ag—Ab), prepared from ¹²⁵I-radiolabelled bovine serum albumin (BSA) and guinea-pig antibody, were (1) pre-incubated at 37°C for 30 min with serum or cerebrospinal fluid (CSF) in different proportions and then reacted with cells, (2) incubated at 37°C directly with cells suspended in serum or CSF for different time periods, or (3) bound to cells (following incubation with serum in optimal proportions) and the cell-bound immune complexes (IC) incubated with serum or CSF at 37°C for different time periods. When Ag—Ab were pre-incubated with serum or CSF and reacted with unfractionated blood cells or mononuclear cells, binding decreased as serum to Ag—Ab proportion was increased above 1:16, but increased as CSF to Ag—Ab proportion was increased. When serum diluted 200-fold (to approximate the protein concentration of CSF) was used in place of undiluted serum, serum-mediated binding paralleled CSF-mediated binding. Inactivation of serum, CSF, and 1:200 serum in different ways and substitution of human red blood cells (RBC) (known to possess C3b receptors) or sheep RBC (known not to possess C3b receptors) demonstrated that binding was to C3b receptors. Addition of CSF to serum did not alter serum-mediated binding. When Ag—Ab were incubated directly with unfractionated blood cells suspended in serum or CSF, binding increased rapidly in serum, reaching a maximum within 2—4 min, and IC then rapidly dissociated, whereas binding increased gradually in CSF and IC remained associated with cells. When serum diluted approximately 100-fold was used in place of undiluted serum, kinetics of serum-mediated interaction approached that of CSF-mediated interaction. When IC were bound to Raji cells or human RBC and the cell-bound IC incubated in serum or CSF, > 85% of IC dissociated in serum after 30 min, but no dissociation occurred in CSF. Dilution of serum > 1:16 and > 1:64 abolished dissociation from the two cell types, respectively. These results indicate that CSF mediates binding of IC to complement receptors on cells but lacks the activities of serum which convert IC into a non-binding state.     

Antigen—antibody complexes and the weal and erythema skin responses

The smallest amount of purified antibody capable of positive weal and erythema response is of the order of 4 μg N.

Antigen—antibody complexes are capable of producing specific weal and erythema reactions with antigen—antibody ratio of 1:1 or above. In these instances antibodies occupied at one or both sites are responsible for this response. Preformed antigen—antibody complexes are capable of mediating this response.

     

The action of soluble antigen—antibody complexes in perfused guinea-pig lung

When soluble antigen—antibody complexes, prepared in antigen excess, were injected into the perfused pulmonary artery of the unsensitized guinea-pig lung, they produced a bronchoconstrictor response which was associated with the appearance of histamine and a slow-reacting substance in the effluent. This activity was inhibited by the presence of normal rabbit serum or rabbit globulin; rabbit albumin and bovine γ-globulin produced no inhibition. Insoluble antigen—antibody complexes prepared at equivalence were at least 125 times less active than soluble complexes prepared in antigen excess. On comparing the activity of different solutions of soluble complex prepared in antigen excess ranging from 2½ to 160 times that required for equivalence, all were found to be equal. The properties of soluble antigen—antibody complexes are consistent with the possibility that circulating antibody may participate in in vivo anaphylaxis, if reached by an amount of antigen greater than that required for equivalence.     

Antibody production in mice: V. The suppressive effect of anti-carrier antibodies on cellular cooperation in the induction of secondary anti-hapten antibody responses

Cooperative induction of secondary anti-hapten antibody responses was studied by using non-cross-reactive carrier proteins, bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole-limpet haemocyanin (KLH), and the 2,4-dinitrophenyl (DNP) and benzylpenicilloyl (BPO) groups as haptenic determinants.

Lymphoid cells obtained from mice primed with a hapten-carrier conjugate could be effectively stimulated with a hapten-heterologous carrier conjugate, provided that lymphoid cells primed to the heterologous carrier were also present. In the carrier-primed lymphoid cell population, helper activity of thymus-derived cells developed earlier following carrier immunization than did the capacity of antibody-forming cell precursors (AFCP) to produce an effective anti-carrier antibody response upon secondary stimulation. Attempts to generate hapten-specific helper cell activity were unsuccessful. Thus, cells primed to one haptenic determinant failed to exert a helper function with cells primed to a second hapten upon subsequent administration to the two haptens together on the same heterologous carrier molecule.

In order to distinguish among carrier determinant specificities which react with thymus-derived helper cells from those which react with bone marrow-derived AFCP, the capacity of various anti-carrier antibodies or antibody fragments to suppress either anti-carrier antibody production alone or together with helper cell function in adoptive secondary anti-hapten antibody responses was tested. In this system, it was found that 7S anti-carrier antibody suppressed the reaction of helper cells and carrier-specific AFCP such that both the anti-hapten and anti-carrier antibody responses were abrogated. By contrast, passively administered 3.5S fragments of anti-carrier antibodies selectively prevented the stimulation of carrier-specific AFCP to produce anti-carrier antibodies, but had no effect on the capacity of carrier-specific helper cells to facilitate the secondary anti-hapten antibody response. As expected, passively administered 7S anti-hapten antibodies selectively abrogated the production of anti-hapten, but not anti-carrier antibodies. These data are discussed in the context of suggesting that distinct determinant sites on carrier molecules are recognized independently by thymus-derived helper cells and by bone marrow-derived AFCP.

     

The effect of antibody on the degradation of a polysaccharide by the reticulo-endothelial system

A galactan isolated from gum arabic was shown to be non-antigenic in mice. However, O-acetylated derivatives of this polysaccharide were antigenic and also gave rise to antibodies which reacted with the galactan.

The degradation of the galactan and its O-acetylated derivatives in vivo by the liver and lungs of normal mice has been followed.

It was found that, whereas, both the liver and lung degraded the galactan, O-acetylated derivatives containing 10–20 per cent by weight O-acetyl groups were degraded only by the lung.

The breakdown of the acetylated polysaccharide by the lung was enhanced by added antibody under conditions where the antigen was in excess. These conditions also led to an enhanced antibody response. In contrast both degradation and antibody formation were inhibited when animals received antigen—antibody complexes in antibody excess. The significance of these results in relation to the anamnestic response is discussed.

     

Characterization of the antibody response to Type III pneumococcal polysaccharide at the cellular level: II. Studies on the relative rate of antibody synthesis and release by antibody-producing cells

A procedure based on the rate of appearance of plaque-forming cells (PFC) in agarose was used to measure the relative rates of antibody synthesis and release by cells making antibody specific for Type III pneumococcal polysaccharide (SSS-III). The rate of antibody synthesis and release by SSS-III-specific PFC was directly related to the immunizing dose employed; maximal values were obtained with mice given an optimally immunogenic dose (0.5 μg) of SSS-III. However, dose-dependent reductions, not only in the magnitude of the antibody response, but also in the rate of antibody synthesis and release by specific PFC, were noted in mice receiving doses greater than 0.5 μg. The latter suggests that a decrease in the rate of antibody synthesis and release by antibody-forming cells may be an initial step in the induction of immunological paralysis by high doses of SSS-III.

Increases in the magnitude of the serum antibody and the PFC response were associated with corresponding increases in the rate of antibody synthesis and release by SSS-III-specific PFC following immunization; this suggests that cell differentiation—rather than proliferation—plays a major rôle in the development of the antibody response to SSS-III. In contrast, the results obtained in similar studies with sheep erythrocytes indicate that cell proliferation influences to a greater degree the magnitude of the antibody response elicited to this antigen.

     

The optical rotatory dispersion of antigen—antibody complexes

The optical rotations of solutions of the antigen—antibody complexes formed by excess of antigen were measured over a range of wave-lengths. The antigens used were bovine serum albumin (BSA) and diphtheria toxin. A method of estimating the optical rotations of the precipitable antibody against BSA in a solution of rabbit γG-globulin was used. The antitoxin used was isolated peptic antitoxin.

The optical rotations found were compared with those anticipated if the optical rotations had not been altered on combination. The optical rotation of the BSA—antibody complex at the D-line did not differ appreciably from that anticipated, but differences were found at shorter wave-lengths.

Mixtures of toxin and peptic antitoxin, in which the molecular ratios of toxin to antitoxin ranged from 2.29 to 0.37, gave rotatory dispersion curves, in the far ultraviolet range, which differed from those of the sums of the separate specific rotations of the constituent toxin, free and combined, and antitoxin. The differences in position and amplitude of peaks and troughs depended on the ratio of toxin to antitoxin. But in every mixture the maximum specific rotation at 205–210 mμ was less than the anticipated maximum rotation. Over the range 230–250 mμ the observed specific rotations of the mixture in which the ratio of toxin to antitoxin was highest were less negative than the anticipated specific rotations; in the mixture in which the ratio of toxin to antitoxin was lowest the observed rotations over the range 225–400 mμ were more negative than those anticipated.

     

Induction of DNA synthesis in normal human lymphocyte cultures by antigen–antibody complexes

Antibodies were produced in various species against particulate (sheep red blood cells and Salmonella bacteria) or soluble (E. coli endotoxin, human serum albumin and mouse γ-globulin) antigens. Antibody mixed with the corresponding antigen stimulated DNA synthesis in normal human lymphocytes cultivated in vitro, whereas antibody alone or antigen alone had no or a negligible effect. The species origin of the antibody did not seem to influence the results. Maximal stimulation of DNA synthesis occurred after 5–6 days. Preformed antigen–antibody complexes were also stimulatory, whereas heat-aggregated γ-globulin had no effect. The addition of fresh complement did not increase DNA stimulation. The degree of stimulation of DNA synthesis after contact between lymphocytes from immunized individuals and the specific antigen was even further increased when humoral antibody was introduced.

The present findings suggest a mechanism by which non-committed lymphoid cells of host origin are activated by a small proportion of antibody-producing cells to participate in various cell-mediated immune reactions, in particular since it was found that lymphocytes stimulated by antigen–antibody complexes acquired the capacity to kill allogeneic and autochthonous fibroblasts.

     

The sheep immune response: Variation of anti-hapten and anti-carrier antibodies localized in the γ1 and γ2 immunoglobulin fractions

The results obtained in the quantification of γ₁ and γ₂ anti-hapten (DNP) and anti-carrier (HGG) antibodies obtained weekly during 50 weeks and proceeding from a group of three Merino sheep immunized with DNP—HGG, are described.

The primary and secondary response and the antibody variations in weekly repeated stimulations were studied.

The anti-DNP and anti-HGG total antibodies have similar variations. γ₁ and γ₂ anti-DNP and anti-HGG antibodies do not show differences in the primary response nor in weekly repeated stimulations.

In the secondary response, a high increase of γ₁ with loss of γ₂ is observed. After a certain time γ₁ decreases and γ₂ increases.

     

Further attempts to characterize the normal incomplete cold antibody

Attempts at eluting the normal incomplete cold (n.i.c.) antibody from sensitized red cells or red cell stroma, using standard methods, were unsuccessful; thus the antibody could not be detected in the eluates obtained by heating at 56° or 37° or by dissociation at acid pH. The n.i.c. antibody was partially eluted from sensitized red cells only when elution was carried out at 37° into serum instead of saline.

Elution of the antibody from dextran (Sephadex G-200)—n.i.c. antibody complex formed at 0° was also achieved using a 15 per cent NaCl solution at 37°.

Since it has been shown that red cells sensitized with the n.i.c. antibody are not agglutinated by anti-γG, anti-γA or anti-γM-globulin sera, an attempt at producing an antibody specifically reacting with the n.i.c. antibody was made by injecting into a rabbit the eluate obtained from zymosan—n.i.c. antibody complex. The immune serum was found to inhibit the n.i.c. antibody activity when added to normal serum and to interfere with the property of normal serum requiring the properdin system.

In the present work it is also confirmed that the antibody is not associated with γG or γM-globulin; thus adult and cord sera and one serum from a patient with severe hypogammaglobulinaemia were fractionated with zone electrophoresis or DEAE-cellulose chromatography and it was found that the antibody was not present in the fractions containing the bulk of γG or γM-globulin.

     

The effects of ALG on the murine immune response to sheep erythrocytes

Antilymphocyte globulin (ALG), and to a lesser extent normal rabbit globulin (NRG), when given to mice prior to immunization with sheep-RBC suppress both the γM and γG_2a responses. Globulin injected after the antigen suppresses the γG_2a response, augments the γG₁ response and has little effect on the γM response. These effects are also observed in mice partially paralysed to rabbit γ globulin. In another system—the response to hapten—protein conjugates precursors of antibody producing cells were found to be more resistant to ALS treatment in vivo than were helper cells. It is concluded that the suppressive effects of ALG treatment are largely due to the direct action of ALG on helper cells (T-cells). The mechanism of the adjuvant-like effect is unclear.     

Antigen in tissues: V. Effect of endotoxin on the fate of, and on the immune response to, serum albumin and to albumin—antibody complexes*

Bacterial endotoxin was injected into rat hind footpads together with bacterial flagellin and ¹²⁵I-labelled human serum albumin (HSA); the latter was used unmodified or heat denatured (H.HSA) or as an HSA—antibody complex. Endotoxin did not affect the trapping, retention nor localization of the labelled HSA in the popliteal and aortic lymph nodes, whether the antigen had been injected as HSA, H.HSA or as an HSA—antibody complex.

If endotoxin was injected at the same time as: (1) flagellin, there was an increased production of anti-flagellin antibody; and (2) H.HSA or HSA—antibody complex, detectable amounts of anti-HSA antibody were produced. When H.HSA and endotoxin were injected, the primary response was long lived yet the period of induction of antibody formation and of antigen persistence in the lymphoid tissues was short. If, during the primary antibody response to H.HSA, the animals were challenged with HSA, equally strong secondary antibody responses occurred with an HSA—antibody complex or with HSA alone.

The results were interpreted in terms of the tissue localization pattern of H.HSA (medullary macrophage) and HSA—antibody complex (medulla and lymphoid follicles). It was suggested that: (1) induction of antibody formation and priming of cells for a secondary antibody response might occur following localization of the antigen in the medulla and that antigen localization in the lymphoid follicles might not be a strict requirement for this; and (2) the follicular localization of antigen might be the preferential mechanism for the firing of a secondary antibody response.

     

Measurement of antigen—antibody complexes in Salmonella gallinarum infection

Following oral infection of a 12-week-old chicken with an overnight broth culture of Salm. gallinarum, serum was removed on the 9th day and tested for the presence of haemagglutinating antibody using erythrocytes modified with a Westphal lipopolysaccharide extract of Salm. gallinarum. The serum was found to have a high titre and on `Sephadex' fractionation and ultracentrifugal preparation showed a marked IgM response.

Spectrophotometric measurements were made of interactions between the O-antigen extract and the whole serum or its component IgM and IgG fractions. Hapten—antibody complexes were measurable with the whole serum and the IgM fraction, but no complexes were detected with the IgG fraction under the conditions of the experiment.

     

Antibody production in mice: I. The analysis of immunological memory

The development of immunological memory was analysed by the use of `in vivo culture technique'. The lymphoid cells from primed mice were transferred into heavily irradiated recipients and the size of memory cell population in primed donor mice was estimated quantitatively by the magnitude of secondary responsiveness.

The production of memory cells was detected about 1 week after the primary administration of antigen, and increased gradually up to approximately 6 weeks. The development of the memory cells carrying information for the synthesis of γG₁-antibodies (γG₁-memory cells) was initiated at the early stage of priming and followed by that of the γG₂-memory cells. Although the ratio of population of γG₂- to γG₁-memory cells changed with lapse of time in primed mice, the original ratio of each population in donor, at any stage after priming, was apparently maintained when cultured in recipients for at least 4 weeks. This indicates that the conversion from γG₁- to γG₂-memory cells does not occur.

From these results, it was suggested that the γG₁- and γG₂-memory cells develop independently in primed mice under the continuous stimulation of primarily administered antigen.

     

Studies on globulin and antibody production in mice thymectomized at birth

Serum globulin levels and antibody against sheep red cells, Salmonella typhi (`O' and `H'), haemocyanin and pneumococcus type 3 capsular polysacharide (SSS III) were studied in C3H/Bi and C57BL×C3H/Bi F₁ hybrid mice thymectomized within 18 hours of birth. Thymectomized mice tend to recover more slowly than intact mice from the physiological hypo-γ-globulinaemia present 3–4 weeks after birth, but the majority of mice that survived beyond 6 weeks had serum globulin levels comparable to those in intact animals. Increased γ_1A-globulin levels were observed in some thymectomized mice which survived for 7 weeks or more and the immuno-electrophoretic patterns sometimes resembled those seen in mice with γ_1A myelomas. Macroglobulin (γ_1M) was detected in the sera of thymectomized mice which contained normal amounts of γ-globulin.

Total serum protein levels of groups of thymectomized and intact mice were not significantly different. The turnover rate of plasma albumin was also closely similar in both groups, but that of γ-globulin was accelerated in most of the thymectomized mice examined.

Antibodies against SSS III, sheep red cell agglutinins and typhoid `H' agglutinins behaved as 7S globulins, in contrast with sheep red cell haemolysins which belonged to the 19S group.

Antibody levels following primary stimulation with sheep red cells and Salmonella antigens were usually, but not always, much lower in thymectomized mice than in intact control mice. However antibody levels against haemocyanin and SSS III were within the range of controls in more than half the thymectomized mice. Few thymectomized mice after stimulation with a mixture of three antigens failed to give a detectable antibody response to all the antigens, and some responded as well as did intact mice.

The significance of these findings is discussed in relation to current theories of the function of the thymus in the development of immune responses.

Many of the older thymectomized mice showed a characteristic wasting syndrome, but it is unlikely that a general failure of antibody response can account for this. Evidence for the presence of auto-antibodies against red cells or nuclear or cytoplasmic constituents was sought but was not found.

     

The secondary anti-erythrocte response of rabbit spleen cells stimulated in vitro

Suspensions of spleen cells from rabbits immunized to sheep erythrocytes and stimulated in vitro by the specific antigen produced high numbers of antibody-synthesizing cells. Several factors were found to affect secondary antigenic stimulation in vitro. The extent of the immune response elicited was a function of antigen concentration. Maximum was obtained by stimulation with 10⁴ to 10⁵ erythrocytes/10⁷ spleen cells; 10⁷ erythrocytes inhibited the response. The maximum usually occurred on days 4–6; glutamine increased the rate at which antibody-synthesizing cells appeared. It was found that the source of the normal serum supplement used in the growth medium markedly affected the results. Storage of rabbit serum reduced its efficacy; after 6 months storage at –10° little or no response was obtained. The addition of specific antiserum on days 1, 2 or 3 reduced the cellular response.

With cells from twenty-six rabbits tested 6–39 months after immunization, maxima of 1700–29,700 antibody-synthesizing cells/10⁷ spleen cells were attained. No relationship was evident between the time from immunization and the extent of the response. The activity of the extracellular antibody produced by the cells correlated roughly with the number of antibody-synthesizing cells. The antibody elicited in vitro was specific and stable on storage at –10° for 2 years. On the basis of sensitivity to 2-mercaptoethanol, it was a macroglobulin. Disc electrophoresis and identification of the haemolytic component showed that the antibody synthesized in vitro migrated with the γ-globulins, at the same rate as the active component in anti-erythrocyte rabbit serum.