Absence of carrier specificity in an adoptive secondary response

Different NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) conjugates were used to study carrier specificity in normal and adoptive secondary responses. When rats were primed with NIP—HSA conjugate and boosted with either NIP—HSA or NIP—CG 45 days later, there was a clear carrier specificity; the homologous carrier caused a stronger response than the heterologous carrier. When the interval between the first and the second injection was extended to 4 months, carrier specificity was much less strict. Adoptive secondary responses were not carrier-specific whether the time between the priming injection and transfer plus restimulation was short (21 days) or long (4 months). Adoptive secondary IgM responses were less dependent on stimulation with the homologous carrier than the simultaneous IgG responses of the same animal.     

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.     

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.

     

The effect of antigen dosage on the response of adoptively transferred cells

Mice were immunized with BSA or HSA in Freund's adjuvant, and their lymph node and spleen cells transplanted into syngeneic hosts, which in most experiments had been irradiated. After transplantation the cells do not synthesize much antibody if left without stimulation, but can be stimulated to do so by injection of BSA or HSA in solution. The response has been studied over a dose range of 10^-3–10⁵ μg. antigen. Stimulation can be detected down to 10^-3 μg. antigen, and reaches a maximum at middling doses. Middling doses stimulate proliferation of the primed cells to an extent which can be measured by ¹³¹IUdR uptake. At high doses both antibody production and IUdR uptake are inhibited. The conclusion is drawn that high concentrations of antigen can paralyse the immunological reaction of primed cells.     

Antigen-induced histamine release from peritoneal mast cells of mice producing reagin-like antibody

The time course of production in the mouse circulation, after different schemes of immunization, of reagin-like and γ₁ antibodies, as measured by the ability of the antisera to induce thermolabile long-latence and thermostable short-latence PCA reactions, has been studied. γ₁ antibody was obtained in all schemes of immunization and its amount in antiserum increased with time. Reagin-like antibody appeared on the 7th day after immunization with sufficient dose of ovalbumin and adjuvant, and on later days with less efficient antigens such as DNP—BSA and DNP—BGG. Maximal amounts of the reagin-like antibody were reached in the early days after immunization and its presence in the circulation had a somewhat transient character; however, it persisted in the circulation for periods of more than 60 days, on occasion.

The time course of antigen-induced histamine release from washed peritoneal mast cells of the immunized mice was also studied. A correlation was observed between the histamine release and the production of reagin-like antibody. It is suggested that the antigen-induced histamine release from washed peritoneal mast cells may be taken as an indication of the production of reagin-like antibody.

Some quantitative aspects of the PCA and histamine release reactions were also studied. PCA reactions were affected similarly by varying either dilution or volume of the antiserum intradermally injected. Histamine release from peritoneal mast cells did not seem to be affected specifically by excess of antigen.

     

In vitro stimulation of spleen cells of the mouse by DNP—carrier complexes

Hapten—carrier complexes were prepared with the 2,4-dinitrophenyl group (DNP) as a hapten and bovine serum albumin (BSA), bovine gamma-globulin (BGG) (heterologous), mouse immunoglobulin (MIg) (isologous) and polyvinyl pyrrolidone (PVP) (thymus-independent) as carriers. All complexes could be used for priming, independent of the type of carrier or the number of DNP groups. No in vitro response, however, could be obtained with any of the DNP—PVP complexes or with the complexes with a low hapten/carrier ratio (about 3). Priming with carriers alone resulted in some in vitro activity on challenge with the homologous DNP—carrier complexes, but this response was less than after priming with one of the homologous DNP—carrier complexes. Cross-reactions between DNP—MIg, DNP—BGG and DNP—BSA complexes could be obtained. In almost all instances in vitro challenge with BSA with the highest number of DNP groups (DNP₂₈—BSA) resulted in the highest activity. Inhibition of the in vitro reaction could to a certain extent be obtained with ε-DNP—L-lysine. These experiments suggest that: (1) for priming, the hapten/carrier ratio is of no importance and the influence of the type of carrier is low; (2) in vitro stimulation can only be obtained with complexes with a high hapten—carrier ratio; when this ratio approaches a maximum (DNP₂₈—BSA) the type of the carrier used by priming seems to be irrelevant; (3) the data from these experiments, from cross-reactions and from inhibition reactions suggest that the stimulating activity of DNP₂₈—BSA is due to the DNP groups, but the activity of complexes such as DNP₁₆—BSA, DNP—BGG and DNP—MIg is at least partly due to the `new antigenic determinant' (NAD) or DNP—NAD groups.     

The production of specific rabbit antibodies by injecting individual antigen—antibody complexes separated from mixed antigens

Injection of a few hundred micrograms of antigen—antibody precipitates of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme, antigen 3 of Pasteurella pestis and myoglobin into rabbits produced a 10–100-fold increase in antibody compared with that injected in the precipitates. Before injection the precipitates had been separated from either ¹³¹I-labelled human serum albumin serological precipitate or fluorescent ovalbumin serological precipitate by the method of Smith, Tozer, Gallop and Scanes (1962b) after the reaction of mixed antigens with mixed antibodies. The antibodies produced by this method precipitated only their homologous antigen from a mixture of it with either ¹³¹I-labelled human serum albumin or fluorescent ovalbumin. If secondary precipitates formed from antibody produced in this way were injected into rabbits in larger quantities, a further 8–35-fold increase in specific antibody was obtained.     

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.     

Cells involved in the in vitro stimulation by DNP—carrier complexes of in vivo primed mouse spleen cells

The target cell of in vitro stimulation of primed spleen cells by hapten—carrier complexes was studied. The antigens DNP₂₈—bovine serum albumin (DNP₂₈—BSA), which gives only 2,4-dinitrophenyl (DNP) specific responses and DNP₁₄—mouse immunoglobulin (DNP₁₄—MIg), which probably reveals DNP as well as carrier specificity (new antigenic determinant) were used. Educated T cells could be stimulated with the antigens used for activation. A similar experiment with educated B cells, however, gave no indication that these B cells could be stimulated with antigen in the absence of T cells. Cortisone treatment of primed mice yielded spleen cells which had a higher activity than spleen cells from unprimed, cortisone-treated mice. This also points to stimulation of T cells by antigen. Treatment of primed spleen cells with anti-thymocyte serum (ATS) and complement (C) abolished stimulation by phytohaemagglutinin (PHA) completely, by lipopolysaccharide (LPS) slightly, while the antigen-specific activity was 90 per cent reduced. This indicates a mainly T cell-specific stimulation by the antigen. A corresponding experiment with anti-plasma cell serum (APCS) and C revealed a complete reduction of LPS activity and a small impairment of the PHA and conavalin A (Con A) activity. However, the antigen-specific activity was reduced by one-third to a half for the different antigens. This is an indication for a specific B-cell stimulation by the antigen, although it is on a lower level than the T-cell stimulation. The role of the hapten in the T-cell stimulation is discussed.     

The influence of epitope density on the immunological properties of hapten—protein conjugates: II. The in vivo and in vitro metabolism of heavily and lightly conjugated protein

In earlier experiments we showed that lightly hapten-coupled bovine serum albumin (e.g. DNP₅—BSA) elicited mainly IgG anti-DNP antibodies and concomitant immunological memory in mice, whereas DNP₅₀—BSA induced a primary IgM response, little IgG antibody and poor memory. Although the latter are characteristic of T cell-independent antibody responses, antibody formation to DNP₅₀—BSA was found to be highly thymus-dependent.

In the present study the metabolism of DNP₅—BSA and DNP₅₀—BSA was investigated. In vivo DNP₅₀—BSA was rapidly cleared from the bloodstream, but persisted at much higher levels in the liver and spleen than DNP₅—BSA. DNP₅₀—BSA was taken up more efficiently by macrophage cultures than DNP₅—BSA, but released comparatively slowly. Analysis of culture supernatants showed that macrophages degrade both antigens to the same degree. We conclude that heavy dinitrophenylation decreases the susceptibility of DNP—BSA to degradation by lysosomal proteases, but the relationship between the metabolic behaviour of various DNP—BSA conjugates and their immunogenic properties is at present unclear.

     

Antigen—antibody complexes in the immune response: I. A analysis of the effectiveness of complexes on the primary antibody response

Soluble crystalline bacterial α-amylase (BαA)-mouse anti-BαA antibody complexes (Ag—Ab complexes) elicited a primary antibody response in mice with a single intravenous injection, while free BαA could not. The response was dose dependent. Ag—Ab complexes were not only phagocytosed but also degraded more rapidly than free BαA in vivo and in vitro but these characteristics themselves were not important for immunogencity of the complexes.

The Ag—Ab complexes phagocytosed by cells in normal spleen and lymph node elicited a primary antibody response when injected into non-irradiated mice but the response was suppressed when anti-BαA antibody was simultaneously injected. On the other hand, free BαA phagocytosed by cells could not elicit the response.

The degraded products of complexes phagocytosed by normal spleen and lymph node cells were highly immunogenic and probably retain antigenic fragments. They elicited an even higher primary antibody response than the original complexes and were also more effective in eliciting a secondary response from primed cells than the original complexes or free BαA. The degraded products of free BαA, however, were ineffective not only for the primary response but also for primed cells.

Ag—Ab complexes prepared with heterologous rabbit antibody were ineffective for the primary antibody response.

     

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.

     

The significance of the protein carrier in the stimulation of DNA synthesis by hapten—protein conjugates in the secondary response

Rabbits were immunized with 2,4-dinitrophenyl (DNP)—protein conjugates. Spleen cell suspensions were prepared and incubated in the presence of various DNP—protein conjugates, the proteins alone, and DNP—lysine. The antigen dependent stimulation of DNA synthesis was used as a measure of the antigenic `activity' of the DNP preparations. It was found that the cells were strongly stimulated by the DNP—protein conjugates used for immunization, and weakly stimulated by the protein alone. Highly substituted DNP—protein conjugates were markedly more effective than lightly substituted conjugates. DNP-conjugates with proteins other than the one used during immunization were inactive. DNP—lysine alone was inactive but inhibited stimulation by the DNP—protein conjugate used for immunization. The significance of these findings is discussed.     

The effect of dose in tolerance induction on the subsequent response to a cross-reactive antigen

The amount of antibody produced by BSA-tolerant rabbits as a result of immunization with DNP—BSA is dependent upon the amount of BSA used to induce tolerance. Tolerance was induced by initial injection of 100 μg of antigen followed by progressively higher doses. Rabbits rendered tolerant with a maximum BSA dose of 1 mg had a mean serum anti-BSA antibody concentration of 0.39 mg/ml after immunization with DNP—BSA, whereas rabbits rendered tolerant with a maximum BSA dose of 100 mg had a mean serum anti-BSA concentration of 0.02 mg after the same course of immunization. This compares with a mean of 1.08 mg of anti-BSA antibody in normal rabbits immunized with DNP—BSA. There was a similar reduction in the concentration of anti-DNP antibodies and of conjugate-specific antibodies in the tolerant groups.

The results are discussed in terms of a thermodynamically-controlled induction of tolerance in individual precursor cells.

     

The carrier effect in the secondary response to hapten-protein conjugates. II. Cellular cooperation

The adoptive secondary response of mice to conjugates of NIP (4-hydroxy-5-iodo-3-nitro-phenacetyl-) and DNP (2,4-dinitrophenyl-) is here used to elucidate the mechanism of cellular cooperation. The framework into which the experiments fit can be formulated as follows. Priming immunization raises a crop not only of specific antibody-forming-cell-precursors (AFCP) but also of specific helper cells. Upon secondary stimulation the helper cells serve a role as handlers or concentrators of antigen, thus enabling AFCP which would otherwise be incapable of reacting to initiate antibody synthesis. In this act of cooperation both cells recognise antigen; in the system examined here the helpers recognise carrier determinants and the AFCP recognise either the hapten or other carrier determinants. The first aim of the experiments was to raise populations of helpers and AFCP of distinguishable specificity. Mice were primed with NIP-Ovalbumin (OA) mixed with chicken γ-globulin (CGG) and bovine serum albumin (BSA); in comparison with controls primed with unmixed NIP-OA, their cells after transfer were relatively more sensitive to secondary stimulation with NIP-CGG or NIP-BSA and similar findings were obtained in cross-checks of these carriers. For reasons which are not entirely clear, non-transferred cells did not show the same effect. In further experiments cells primed with one conjugate (e. g. NIP-OA) were mixed with cells primed with another protein (e. g. BSA), transferred and challenged with the hapten conjugated to the second protein (i. e. NIP-BSA). In comparison with controls lacking the protein-primed cells, the mixture regularly showed greater sensitivity to stimulation. NIP and DNP were tested in many of the possible combinations with BSA, OA and CGG with the same result. The mixture system was used in the further analysis. Tests with allotype-marked protein-primed cells showed that these cells did not participate in the production of the anti-hapten antibody and could therefore properly be regarded as helpers. Tests of specificity showed that physical union of the hapten and carrier were required: cells primed with BSA, for example, would not help NIP-OA-primed cells to make a response to NIP-HSA even when stimulated at the same time with BSA. Transfer of less than one-tenth of the spleen gives a maximum helper effect, whereas AFCP activity continues to rise as larger numbers of cells are transferred. Helper cells are therefore normally present in excess. Helper activity is more resistant than AFCP activity to irradiation, drugs and semi-allogeneic cell transfer across an H-2 barrier. This suggests that helper cells play a relatively passive role in the immune response. Several observations indicate that helper cells are thymus-derived mediators of cellular immunity. Passively transferred antibody did not substitute for helper cells. After immunization helper activity developed faster than AFCP activity. Spleen cells obtained from lethally-irradiated, thymocyte-repopulated, immunized donors provided help. Cells from the thymus-derived fraction of thymus/marrow chimeras also appear to provide help. Thus, the hapten-carrier cooperative response maps onto the well-established synergy of thymus and marrow in the response to foreign erythrocytes.     

Inhibition of delayed hypersensitivity by pre-immunization without complete adjuvant

Guinea-pigs were immunized by injections of blood group substance with incomplete adjuvant, followed after an interval of approximately 2 weeks, by intracutaneous immunization with the same antigen and Freund's adjuvant containing M. tuberculosis. This treatment inhibited the appearance of delayed skin reactions, while circulating antibody production took place as in controls which had received complete adjuvant only with blood group substance, and had delayed skin reactions. The inhibition of the skin reaction was found to be antigen-specific with regard to unrelated antigens, but showed cross-inhibition for serologically different human blood group substances. The first immunization had to be given more than 2 days before the immunization with complete adjuvant. A similar phenomenon was seen with ovalbumin as antigen. In addition to inhibition of the delayed skin reaction, there appeared to be less γ₂-antibody to ovalbumin than in ovalbumin plus complete adjuvant-only controls. Passive administration of antibody did not affect the development of a delayed hypersensitivity state in complete adjuvant-immunized animals with blood group substance or ovalbumin as antigen. Present evidence favours an explanation of the phenomenon in terms of temporary paralysis on the part of some of the antibody-producing cells—viz. those concerned with delayed hypersensitivity and γ₂-antibody production.     

Secondary response to bovine serum albumin in diffusion chambers: Stimulation with low doses of antigen

Mice were primed by footpad inoculation of small amounts of BSA (0.1–2.5 μg) in incomplete Freund's adjuvant. Lymphoid cells removed from these mice 50–120 days after priming were cultured in diffusion chambers, either in the presence of BSA or following a brief period of incubation with BSA in vitro. Small but clearly measurable amounts of anti-BSA antibody were produced by cells stimulated with amounts of antigen in the range 0.1–10 μg. The optimum boosting dose seemed to be related to the priming dose. The system described appears to be eminently suitable for the autoradiographic follow up of the interaction of cells with antigen in the secondary response.     

β Toxin of Clostridium welchii : Cytotoxic Effects Produced by the • on Guinea-Pig Monocytes

The cytopathic effects of Cl. welchii β toxin on guinea-pig monocytes in vitro have been studied using the uptake of eosin-Y^* as evidence of cell death.

The experiments show that these effects are due to antigen—antibody reaction probably on the cell surface, and that these reactions are not dependent on the presence of complement. Monocytes can be actively sensitized in vivo, and passively sensitized in vitro. The serum used to passively sensitize the monocytes need not possess a precipitating antitoxin titre.

Comparable experiments using an ovalbumin antigen—antibody system produced the same cytopathic effects on the monocytes as those which occurred in the β toxin—cellular system.

     

Isolation and characterization of immunoferritin conjugates: I. The molecular ratio

¹²⁵I-labelled γ-globulin was conjugated to ferritin for the determination of the molecular ratio in ferritin—γ-globulin conjugates. Equimolar ratios were found in all conjugates free of uncoupled γ-globulin.     

Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS-OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS-OVA primed potent antigen-specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4⁺ T cells. CD8⁺ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)-2. Primed popliteal lymph node cells produced IL-2, IL-5 and interferon (IFN)-γ, but not IL-4 when restimulated with OVA in vitro. Serum antigen-specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS-OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS-OVA also primed local antigen-specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4⁺ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.