In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

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.

     

Induction and Maintenance of the Antibody Response by Different forms of Human Serum Albumin

Various forms of human serum albumin (HSA) were compared in their ability to induce and maintain the antibody response. In an in vitro model system, antibody synthesis was induced spontaneously during the maintenance phase of the immune response–presumably by persisting antigen. When different lymph nodes (LN) of the same rabbit were primed simultaneously with different forms of HSA, the spontaneous responses obtained in cell cultures prepared from LN primed with high molecular weight forms of HSA were greater than the responses obtained in cell cultures prepared from similar LN primed with lower molecular weight forms of HSA. This difference in response was consistent regardless of the method employed in antigen preparation and persisted for many months. The in vitro results indicating that the antibody response would be maintained at a higher level in animals immunized with high molecular weight forms of antigen and that the method of preparation would not be of major significance were confirmed in vivo in a mouse system.     

Study of the cross-reaction between rabbit anti-bovine serum albumin antibodies and equine serum albumin

Cross-reactions between bovine serum albumin and equine serum albumin were studied using heterologous soluble complexes and specifically purified cross-reacting antibody.

Experiments with soluble complexes showed that homologous antigen can displace heterologous antigen specifically bound to antibody but heterologous antigen cannot displace homologous antigen. On gel precipitation tests a specific precipitation resulted when heterologous soluble complex reacted with homologous antigen.

By using equine serum albumin conjugated to polyaminopolystyrene the cross-reacting antibodies from anti-bovine serum albumin imune sera could be isolated. These are divalent 7S, γ-globulin antibodies. A figure of cross-reaction was obtained when these purified antibodies were tested by double diffusion in agar with bovine and equine serum albumins.

The results obtained both with soluble complexes and with purified antibody support the view that cross-reacting antibody is more avid for the homologous than for the heterologous antigen.

     

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.

     

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.

     

Suppressor cell induction in vitro. I. Kinetics of induction of antigen-specific suppressor cells.

The induction of antigen-specific suppressor cells in vitro, using high concentrations (100 mug/ml) of keyhole limpet hemocyanin (KLH) in Marbrook flasks is described. Spleen and cortisone-resistant thymocytes were the richest source of suppressor cell precursors, compared to lymph node cells, peripheral blood lymphocytes or thoracic duct lymphocytes. Suppressor cells induced with KLH only suppressed KLH-reactive helper cells, and not B cells or helper cells of other specificity. The suppressor cells were T cells, as judged by their sensitivity to anti-Thy-1.2, heterologous anti-T, but not anti-B antisera.     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

In vitro studies of `antigenic competition': II. Reconstitution of the immune defect and the relationship between antigen-induced suppression and non-specific enhancement

The experiments described in this paper extend the observations of previous work in vitro demonstrating a decrease in the frequency of antigen-reactive units specific for horse RBC in spleen cells from mice primed with KLH. It was observed that the addition of lymphoid cells having T-cell function reconstituted the anti-HRBC response to normal values. Significant enhancement of the response beyond the normal values could be evoked using two dissimilar methods, (i) addition of allogeneic thymus cells, and (ii) restimulation in vitro with the priming antigen. The latter type enhancement was elicited by the addition of small doses of KLH to cultures of spleen cells from mice recently primed with KLH, including cultures otherwise demonstrating antigen-induced suppression. The stimulus required to enhance the response to HRBC in these cultures was specific for the priming antigen, KLH. These results are discussed in the light of current theories of `antigenic competition' and specific heterologous enhancement.     

The suppressive effect of carrier priming on the response to a hapten-carrier conjugate

Carrier-primed cells, having some of the properties of T lymphocytes, have been found to inhibit the primary anti-hapten response of spleen cells to challenge with a hapten carrier conjugate. The antibody response of CBA/Ig^b spleen cells in irradiated CBA (Ig^a) recipients was measured by means of a radioimmunoassay using ¹²⁵I-labeled anti-Ig^b antibody. It was found that the primary anti-2,4,6-trinitrophenyl(TNP) response to TNP-KLH (keyhole limpet hemocyanin) was suppressed in recipient mice primed with KLH as compared with similarly treated unprimed or bovine serum albumin-primed recipients. This suppression was transferred by injection of 10⁸ spleen cells from CBA mice primed with KLH seven days beforehand, but not by injection of serum from such mice. The suppressive effect was abolished by treating the carrier-primed spleen cells with anti-T cell sera and complement before transfer. Priming with KLH seven days beforehand had only a small suppressive effect on the response to an unrelated antigen, DNP-ovalbumin, but there was marked suppression if KLH was again administered with the unrelated antigen. It is considered that the suppressive effect is specific in induction, but nonspecific in expression and that it is a manifestation of a homeostatic mechanism limiting the extent of the immune response.     

The localization of non-microbial antigens in the draining lymph nodes of tolerant, normal and primed rabbits

Soluble haemocyanin (HCy) or human serum albumin (HSA) labelled with ¹²⁵I at 8 and 0·7 μc/μg, respectively, were injected into the footpads of rabbits in doses just sufficient to elicit a primary response in normal animals, and the distribution of radioactivity in the popliteal lymph nodes between 6 hours and 21 days later was studied by autoradiography. The recipient rabbits had either been primed by a single prior injection of unlabelled antigen, or made putatively tolerant by repeated neonatal administration of antigen, or were previously untreated. Localization of antigen in germinal centres, in a typical dendritic pattern, was marked in the primed animals throughout the period of observation; in those tolerant animals which had no detectable serum antibody initially and made no detectable antibody response such localization was not seen at any time; in the animals that had no previous contact with antigen there was no localization in germinal centres until about the time when antibody became detectable in the serum. Localization of radioactivity in medullary sinus macrophages did not differ significantly between the three groups.

It is concluded that localization of these soluble antigens on the dendritic web in lymphoid follicles occurs as a consequence of the presence of circulating antibody. Uptake of the antigens by medullary macrophages, however, can occur in the absence of antibody. Although the degree of labelling of medullary macrophages was not evidently affected by the presence of antibody in these experiments, it is emphasized that the antibody levels, even in the primed animals, were low, and that this finding is unlikely to apply when the amount of antibody present is relatively much greater than the amount of antigen injected.

     

Persisting T cells in rats tolerant of human serum albumin. The significance of tolerant and nonimmune T cells which preferentially restrict high affinity antibody synthesis

The adoptive response of primed rat thoracic duct lymphocytes (′TDL) following specific antigen challenge (soluble human serum albumin, s-HSA) was restricted when cells were transferred into syngeneic, adult (AS2 × AS) F₁ hybrid recipients in comparison with irradiated hosts. This adoptive memory response was also inhibited in irradiated recipients by transferring nonimmune TDL along with ′TDL. Recirculating B cells (B-TDL) did not inhibit the ′TDL response, indicating that the adoptive secondary response was regulated by T cells. Antibody synthesis was preferentially restricted in the high affinity memory cell precursor population, demonstrating a role for T cells in regulating the maturation of antibody affinity. The adoptive memory response was liberated from this T regulatory effect in adult recipients when hosts were challenged with the alum-precipitated adjuvant form (HSA-adj) rather than the soluble form of HSA. Since the adoptive memory response was sensitive to the presence or absence of T cells, this experimental model was used to determine whether or not T cells were eliminated from HSA-tolerant rats. Antibody synthesis by ′TDL was reduced approximatel 10-fold compared with controls when transferred into tolerant recipients and challenged with either s-HSA or HSA-adj; a similar reduction was not observed by substituting bovine serum albumin (BSA) ′TDL and challenging with s-BSA. The tolerance-induced inhibition of HSA ′TDL was destroyed by irradiation and TDL from HSA-tolerant donors were more effective than normal nonimmune TDL in reducing the adoptive HSA ′TDL response. HSA-tolerant TDL did not inhibit the BSA ′TDL response significantly. The results indicate that T cells are not eliminated by tolerance induction in this model and after interaction with tolerogen may exert an activ (or competitive) role in restricting antibody synthesis by high affinity B memory cell prcursors. However, the fact that tolerant T cells are not able to prevent a primary response suggests that unresponsiveness to HSA in the T compartment represents a functional deficiency and not an active suppression at this level. Nevertheless, the presence of these tolerant cells probably accounts for the failure of antibody affinity to mature in partially tolerant rats.     

Antigen in tissues: IV. The effect of antibody on the retention and localization of antigen in rat lymph nodes*

A study has been made of the role of specific antibody in causing increased retention and specific localization of a weak antigen, human serum albumin (HSA), in the popliteal and aortic lymph nodes of rats. The antigen was labelled with ¹²⁵I prior to mixing with antibody. HSA mixed with excess homologous antibody was trapped to the greatest extent in these nodes after footpad injection of the antigen. Injection of HSA with antibody caused increased uptake of HSA into the medulla but retention was poor as autoradiographs showed the area to be essentially free of antigen 4–5 days after injection. By contrast, antigen injected with antibody localized strongly in lymphoid follicles and persisted at this site. Both IgM and IgG antibody were found to cause follicular localization of HSA in rats. Heterologous, isologous and homologous antibody also caused follicular localization of the antigen. Purified homologous γ-globulin was found to localize in the follicles. A moderate increase in the net negative charge of the γ-globulin by acetylation did not appreciably affect the ability of the globulin to localize in the follicles. Detectable formation of antibody did not occur in the rats after injection of antigen—antibody complexes, owing possibly to the inhibitory effect of free antibody on the primary response.     

Recall of antibody synthesis to the primary antigen following successive immunization with heterologous albumins. A two-cell theory of the original antigenic sin

Chickens primed with human serum albumin (HSA) and challenged with duck serum albumin (DSA) will recall antibody synthesis specific for the first antigen. There was a direct relationship between the antigen dose requirements for priming and challenge. Equal avidity of anti-HSA antibodies following challenge with HSA or DSA was found. Immunization with HSA did not enhance the response to challenge with DSA. Challenge with DSA of chickens tolerant to the priming antigen (HSA) was followed by a marginal and transient “termination” of tolerance. Tolerance to the challenging antigen (DSA) did not interfere with the recall of anti-HSA antibody synthesis in HSA-primed chickens. Several structurally unrelated antigens did not manifest the recall effect. In view of the lack of serological cross-reactivity between DSA and HSA, the mandatory role in the “recall phenomenon” is ascribed to T lymphocytes which react with a common epitope of both albumins.     

Induction of Autoimmunity to Adrenal Gland

Rabbits that were immunized with homologous and heterologous adrenal homogenates in complete adjuvant responded with complement-fixing heat-stable autoantibody primarily directed against adrenal. Rabbits responded with autoantibody production against cytoplasm of adrenal cortex and of ovarian and testicular cells as detected by immunofluorescence. Antibody, with sedimentation similar to 7S γ globulin, was directed against a heat-labile antigen in rabbit, guinea-pig and rat adrenal. Antibody responsible for reactions with the antigens in the complement fixation and immunofluorescent tests was absorbed by an ultra-centrifugal sediment of adrenal or ovary.

Histological evidence of adrenalitis was present only in those rabbits immunized with heterologous adrenal.

Guinea-pigs immunized with heterologous adrenal homogenates developed more extensive adrenal infiltrates than did guinea-pigs immunized with homologous adrenal. Antibody from guinea-pigs immunized with heterologous adrenal reacted with autologous organ antigens as well as with rabbit and rat adrenal. Skin tests with homologous adrenal revealed no evidence for delayed-type hypersensitivity.

The detection of autoantigens in foetal rabbit adrenal and ovary suggests that rabbits should be tolerant to such antigens. The greater efficacy of immunization with heterologous adrenal in eliciting autoantibody and autoimmune adrenalitis may be a feature of experimental autoimmunity induced by those organ-specific antigens to which the animal is tolerant. The presence of autoantibody in the serum, bound γ globulin at the site of adrenal lesions and the absence of delayed-type hypersensitivity to homologous adrenal suggest that the humoral immune mechanisms play a role in experimental autoimmune adrenalitis.

     

T cell-dependent mediator in the immune response: II. Physical and biological properties

Supernatants were obtained from cultures of keyhole limpet haemocyanin (KLH) primed mouse spleen cells, and also from cultures of KLH-educated, thymus-derived lymphocytes (T cells) after re-exposure of cells to the original priming antigen, KLH.

These supernatants were able to facilitate the immune response of bone marrow-derived lymphocytes (B cells) to sheep erythrocytes (SRBC).

Certain physical and biological properties of such supernatant activities are described.

     

Monitoring of CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis

CFSE dye dilution analysis and [³H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [³H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7–53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4⁺ KLH-reactive T cells but also revealed a substantial population of CD8⁺ KLH-reactive T cells in one patient as well as minor populations of CD8⁺ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.     

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—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.

     

Cellular events in protein tolerant inbred rats. I. The fate of thoracic duct lymphocytes and memory cells during tolerance induction to human serum albumin

(AS2 x AS)F₁ hybrid rats were made completely or partially tolerant to an adjuvant challenge of human serum albumin (HSA) by prior treatment with fluid HSA (tolerogen). Single doses (10 μg to 200 mg) of fluid HSA failed to elicit a detectable immune response. However, rats made partially tolerant by repeated small doses (10 – 100 μg) synthesized significant quantities of antibody in response to the treatment. Completely unresponsive rats (100 mg fluid HSA 3 x/week for 8 weeks) contained no detectable antigen-binding antibody in their serum and no plaque-forming cells (PFC) in their spleens. Thoracic duct lymphocytes from these animals failed to respond when adoptively transferred to irradiated, bone marrow restored recipients and did not inhibit the response of equal numbers of normal thoracic duct cells. Normal lymphocytes responded normally in completely tolerant rats, whether the recipients were irradiated or not. Cells from the bone marrow of tolerant rats were able to restore HSA responsiveness to irradiated rats within weeks of repopulation. Partial tolerance was characterized by a reduction in PFC per spleen, a diminished antigen-binding capacity (ABC) in the serum, a rapid decline in ABC with time and inhibition or abolition of a memory response. Analysis of the dosage requirements for HSA-tolerance failed to support the concept that tolerance was induced at two separate dosage thresholds of antigen, i.e. “high” and “low” zone tolerance. To the contrary, the degree of unresponsiveness increased continuously in proportion to the dose of antigen and the number of injections, implying that tolerance induction can be explained by a single mechanism at all doses.