Membrane and metabolic requirements for tolerance induction of neonatal B cells

The metabolic requirements of tolerance induction of immature B cells has been analyzed through the use of various putative inhibitors. The study utilizes the splenic fragment assay in which tolerance induction of individual B cells can be examined. Concentrations of inhibitors were determined which, if removed after the first 18 hr of culture, before antigenic stimulation, had no inhibitory effects. Thus, by adding tolerogen in the presence or absence of inhibitor during the first 18 hr of culture, the effect of that inhibitor on tolerance could be assessed. By using this protocol, the data indicate that several metabolic functions of the cell are necessary for tolerance induction to occur, including RNA biosynthesis, DNA biosynthesis, and a methyltransferase reaction, because drugs that interfere with these metabolic processes also prevent tolerance induction. Our previous studies indicated that protein biosynthesis and energy generation are also required. However, drugs that interact with the cytoskeletal structure of the cell and inhibit surface immunoglobulin capping do not interfere with tolerance induction. Moreover, colchicine, which inhibits cell division, does not inhibit B cell tolerance. Collectively, the results provide compelling evidence that the mechanism of immature B cell tolerance involves an active process requiring several metabolic activities of the cell.     

Hapten-specific murine colony-forming B cells. II. Delineation of a tolerogen-sensitive subpopulation of colony-forming B cells

B cell subpopulations were studied by using B cell cloning procedures and an in vitro tolerance induction model. Fluorescein- (FL) specific B cells from normal spleens were isolated by using FL gelatin plates and were then cultured in semisolid agar in the presence or absence of tolerogen. Hapten-specific cells grew in soft agar to form discrete colonies. Colony growth is dependent on "mitogens" present in agar, sheep red blood cells (SRBC), and lipopolysaccharide (LPS). For example, SRBC plus LPS potentiate the growth of an increased number of colony-forming B cells (CFU-B) compared to either additive alone. These CFU-B could be triggered by a specific antigen to yield plaque-forming cells (PFC). With tolerogen (FL-sheep gamma-globulin) present in the agar, the number of FL-specific CFU-B was reduced by 25 to 50%. The ability of the remaining colonies to form PFC upon antigenic stimulation was also reduced. This reduction in CFU-B numbers, however, was observed only when the agar contained both SRBC and LPS as mitogenic potentiators of growth; no effect of tolerogen on CFU-B numbers was seen when cells were grown with either additive alone. Interestingly, the effect of tolerogen on CFU-B numbers was abrogated when peritoneal macrophages, in addition to SRBC plus LPS, were present during cloning. It is postulated that unique subpopulations of B cells form colonies under varied cloning conditions and that those CFU-B grown with SRBC plus LPS display an increased sensitivity to growth inhibition by tolerogen.     

Establishment of unresponsiveness in primed B lymphocytes in vivo

As an approach to examine the influence of the state of cellular activation on the ability to tolerize B cells, the induction of unresponsiveness in human gamma-globulin-(HGG) primed B lymphocytes was studied in an adoptive transfer system. In contrast to transferred normal spleen cells, spleen cells from HGG-primed mice are not readily rendered unresponsive when exposed to the tolerogen, deaggregated HGG (DHGG), in irradiated recipients. A kinetic study showed that unfractionated primed spleen cells do not respond to an antigenic challenge given between 6 and 10 days after cell transfer and injection of DHGG, indicating that they are transiently depressed. In contrast, isolated primed B cells are tolerized when transferred to recipients and treated with DHGG in the absence of T cells. Furthermore, primed B cells exposed to tolerogen in the recipients do not recover the ability to respond to HGG either after a secondary challenge with AHGG given up to 14 days after transfer, or after 2 consecutive challenges given on days 14 and 24 after transfer. The presence of primed T cells at the time of tolerization interferes with the induction of unresponsiveness in these primed B cells. These studies suggest that the presence of primed T cells is responsible for the inability to tolerize unfractionated primed spleen cells populations and that primed B cells themselves are not intrinsically resistant to the induction of unresponsiveness.     

T cell tolerization in vitro: modulation of helper and suppressor cell activities

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.     

Effect of priming with a thymus-independent antigen on susceptibility to B-cell tolerance.

The effects of priming on the susceptibility of B-cell subsets to tolerance induction have been tested in a model system in which anti-immunoglobulin (anti-Ig) has been employed as a surrogate for tolerogen. T-cell-depleted B cells were primed in vitro with fluorescein or trinitrophenylated Ficoll (a thymus-independent (TI) antigen) and then exposed overnight to anti-Ig to attempt to induce B-cell anergy. Primed cells were relatively resistant to this tolerance protocol and resistance was hapten specific. The dose response and kinetics suggested that this process was not due to receptor blockade or modulation, but was an active process. Moreover, this priming for resistance to tolerance was reproduced in vivo upon intraperitoneal treatment with haptenated Ficoll. Such in vivo priming for tolerance resistance was long-lasting and did not occur with a thymus-dependent priming protocol with fluoresceinated hemocyanin. These results are discussed in terms of TI priming to drive B cells into cycle and express novel functional and phenotypic properties.     

Interference with tolerance induction of primed B cells by the Fc fragment of immunoglobulin

The capacity to interfere with tolerance induction in primed B cells was examined. Previous work had shown that TNP-specific splenic B cells from mice primed and boosted with TNP-KLH are highly susceptible to in vitro tolerization upon a brief exposure to TNP on a carrier unrelated to KLH. In the present work it was found that tolerance induction in these primed B cells could be partially disrupted by addition of the Fc fragment of immunoglobulin, a B-cell mitogen, and adjuvant, during exposure of the B cells to tolerogen. Addition of Fc fragments prepared by papain digestion of human IgG interfered with tolerization routinely in approximately 30-60% of the spleen cells susceptible to tolerogen. Addition of whole IgG or Fab fragments had no effect on tolerance induction. As little as 5 micrograms/ml of the Fc fragment preparation significantly interfered with tolerization and 32-64 micrograms/ml was optimal. Disruption of tolerization was most effective when the Fc fragment was added to the spleen cells either 4 hr prior to tolerogen or simultaneously with tolerogen; addition of the Fc fragment 4 hr after exposure to tolerogen was significantly less effective. Disruption of tolerization by the Fc fragment was not through polyclonal activation of B cells, as antigen was required for generation of significant numbers of PFC to TNP. Also, disruption was not through expansion of low avidity clones of B cells insusceptible to tolerogen, as the avidity of the antibody produced with and without Fc fragments present was approximately the same. These results show that the Fc fragment of IgG can partially interfere with tolerization of primed B cells. The manner in which Fc fragments may function to prevent tolerization through its lymphoid cell stimulatory capacities is discussed.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. I. Organ differences of tolerogen susceptibility and cellular sites responsible for the resistance.

Cellular sites of the tolerogen resistance in DDD mice against human IgG (HGG) were examined by reconstitution experiments in which cells of various lymphoid organs from tolerized mice were transferred into lethally irradiated syngeneic recipients with or without the supplement of an excess number of untreated T or B cells. It was shown that T cells but not B cells in the spleen and bone marrow-locating B cells were tolerogen resistant. Kinetic profiles of tolerance induction were compared among thymus, lymph node, and spleen T cells. Thymus cells fall into unresponsive state as early as 2 days after the tolerogen (tHGG) injection when only partial tolerance was observed in lymph node T cells. By 1 week of tolerogen treatment, the tolerant state was completed in both thymus cells and lymph node T cells, while spleen T cells showed marked resistance. Tolerance induced in thymus cells and spleen T cells was of relatively short duration and responsiveness was completely recovered by 5 weeks after the injection of tHGG. At this time lymph node T cells still showed hyporesponsiveness. The differences in tolerance inducibility were also shown among different lymphoid organs in tolerogen dose response. Lymph node T cells were very sensitive to tolerance induction, giving no response even by the injection of 0.01 mg of tHGG. Thymus cells were much less sensitive with the gradual loss of responsiveness by increasing the amount of tHGG. In contrast, spleen T cells showed gradual resistance with increasing amount of tHGG, indicating that some positive response was evoked in spleen T cells by a relatively high dose of tHGG. These results seem to suggest that the tolerogen resistance of spleen T cells may be due to their capability of showing positive response against the tolerogenic material. This was also suggested by the fact that the treatment with cyclophosphamide following the tolerogen injection diminished completely the responsiveness against the subsequent challenge immunization.     

Tolerance and contact sensitivity to DNFB in mice. VII. Functional demonstration of cell-associated tolerogen in lymph node cell populations containing specific suppressor cells.

Adoptive tolerance to contact sensitivity to DNFB is mediated by suppressor T cells. These cells are induced by iv injection of the hapten DNB-SO₃. Experiments were carried out to investigate the question of simultaneous transfer of tolerogen (DNB-SO₃ or its conjugation product DNP) with the suppressor cells. The results showed that tolerant lymph node cells pretreated in vitro with anti-TNP serum before transfer were unable to induce unresponsiveness to DNFB. Tolerant cells treated with either anti-TNP serum which had been passed over a TNP-affinity column or with polyvalent anti-immunoglobul in serum were not inhibited. These results functionally demonstrate that LN cell populations containing DNFB suppressor cells have accessible hapten (e.g., DNP) associated with their membrane, which is necessary for induction of adoptive tolerance. The hapten (tolerogen) appears to be bound directly to the cell surface rather than as an immune complex.     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

Influence of cortisone on immunologic tolerance in vivo: Cortisone prolongs the duration of unresponsiveness but fails to affect its induction

The influence of cortisone administration on either the induction or the duration of immunologic tolerance was examined in vivo. Tolerance induced by isologous IgG coupled to fluorescein was chosen because the hapten-bearing cell can be directly visualized and the hapten-specific immune response to either a TD antigen or a TI₂ antigen can be tested. It was found that cortisone facilitates the maintenance of tolerance, but fails to affect its induction to either class of antigen. Fluorescein-IgG-bearing cells are cortisone resistant. They are seen for a longer period of time in animals treated with cortisone and tolerogen than in animals treated with tolerogen, and fluorescent cells are either T or B cells. We propose that cortisone facilitates the maintenance of tolerance by maintaining a receptor blockade in vivo. This finding might have clinical implications for the treatment of autoimmunity.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. V. Evidence for b-cell deletion in vitro with serum from tolerant mice.

The serum from mice that had been rendered specifically tolerant (TolS) to the trinitrophenyl (TNP) hapten by the injection of trinitrobenzenesulfonic acid (TNBS) is effective in the in vitro induction of immunological unresponsiveness in murine spleen cells. This tolerance system was investigated with particular emphasis upon the mode of induction. The observed inhibition by TolS of responses to the thymic-independent (TI) antigen TNP-lipopolysaccharide (TNP-LPS) was stable following adoptive transfer to lethally irradiated recipients and was due neither to the delay of in vitro responsiveness nor to effector cell blockade at the level of the antibody-forming cell. Neither suppressor cells nor cell-bound tolerogen carry-over were responsible for the tolerance induced by TolS. TNP-LPS doses, including a wide range of polyclonal activating concentrations, were ineffective in reversing the unresponsive state induced by cocultivation with TolS. Additionally, unconjugated LPS in either fetal calf serum (FCS)-containing or FCS-free cultures did not break tolerance. This failure of polyclonal activating substances to reverse the unresponsive state suggests that blockade of TNP-specific receptors is not the mechanism of tolerogenesis, since such compounds trigger cells polyclonally through nonimmunoglobulin receptors. Tolerance induced by incubation of spleen cells with TolS for 24 hr followed by extensive washing was stable whether the immunogenic stimulus was the TI antigen TNP-LPS or the thymic-dependent (TD) form of the hapten, TNP-sheep erythrocytes (TNP-SRC). Washing spleen cells at elevated temperatures after preculturing with TolS to avoid possible reassociation of surface Ig (sIg)-bound TNP conjugates did not lead to escape from tolerance. Antigen-free incubation for 24 hr following cultivation with TolS was equally unsuccessful in reversing the unresponsive state. Thus, extensive washing following tolerance induction and antigen-free cultivation where unblocking or turnover and resynthesis of sIg receptors should have taken place provided no support for receptor blockade as the mode of in vitro induction and maintenance of tolerance by TolS. Treatment with the proteolytic enzyme pronase with the intention of removing potential tolerogen from the cell surface revealed a stable tolerant state. Incubation with anti-Ig or anti-TNP antisera under conditions designed to allow capping and removal of sIg-bound tolerogen or surface-bound TNP conjugates also failed to reverse the tolerance induced by incubation with TolS. The results presented here and previously lend no support to active or passive suppression or blockade of reactive cells as the mechanism of tolerance induction in vitro by TolS. The data are consistent with the hypothesis that TolS-induced unresponsiveness is due to a functional deletion of TNP-specific B lymphocytes. Furthermore, the similarities observed between the induction of tolerance by TNBS injection and TolS-induced unresponsiveness are consistent with the suggestion that TNBS-induced tolerance in vivo is mediated by a component of TolS which is active as a tolerogen in vitro.     

Membrane defects of the tolerant B cell. I. Failure of antigen-induced capping.

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub (J. Immunol.112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

Preventive effect of hapten-reactive thymus-derived helper lymphocytes on the tolerance induction in hapten-specific precursors of antibody-forming cells.

The role(s) of helper T lymphocytes in preventing or altering tolerance induction in DNP-specific B lymphocytes was studied. As DNP-reactive helper T cells were reactive against the DNP-portion of the DNP-D-GL molecule, we could probe definitively the physiological role of helper T cells in preventing tolerance induction in B lymphocytes by DNP-D-GL. The results demonstrated that the induction of DNP-specific B cell tolerance by DNP-D-GL can be completely prevented by the presence of DNP-reactive helper T cells, and provide evidence that one critical role of helper T cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T cell function, might otherwise ensue after binding of the antigenic determinants by specific B lymphocytes.     

The affinity threshold for antigenic triggering differs for tolerance susceptible immature precursors vs mature primary B cells

The specificity of antibody responses is dependent on the extent to which a given antigen selectively stimulates cells from within a diverse B cell repertoire. Previous studies have shown that the triggering of B cells by T cell-dependent antigens is a highly discriminatory process, and that tolerance induction of immature B cells by antigen is equally discriminatory. This symmetry in the requirements for stimulation and tolerance induction could provide a basis for the capacity of antibody responses to discriminate among foreign antigens and yet minimize self recognition. The extent to which this potential for discriminate recognition is applicable to the mature immune system remains controversial, because B cells reactive to self antigens have been identified and, in addition, several investigators have identified heteroclitic immune responses, such as the response to NP of Ighb mice, wherein antibodies are found with higher affinities for analogues of the immunogen than for the immunogen itself. To further investigate the capacity of B cells to discriminate among closely related antigenic determinants, we analyzed the fine specificity and idiotypic distribution of monoclonal antibodies derived from both splenic B cells and immature sIg- bone marrow B cell precursors stimulated in fragment culture with NP-Hy and its structural analogues NIP-Hy and NNP-Hy. The results indicate that the majority of responsive B cells discriminate among these haptenic determinants; however, lambda-bearing B cells responsive to the NP and NIP determinants represent a highly overlapping set of clonotypes. Comparison of the responses to NP-Hy and NIP-Hy of splenic vs sIg- precursors of this clonotype family suggests that the T cell-dependent stimulation of both mature and immature B cells by antigen is highly affinity dependent. Significantly, the affinity thresholds for both stimulation and tolerance induction of immature B cells appears to be higher than that required for the stimulation of mature splenic B cells. Such a disparity in the requisites for triggering mature vs immature B cells could readily account for the presence of low-affinity self-reactive B cells in the mature B cell pools of normal individuals.     

Tissue-resident memory CD8+ T cells can be deleted by soluble, but not cross-presented antigen

Under noninflammatory conditions, both naive and central memory CD8 T cells can be eliminated in the periphery with either soluble peptide or cross-presented Ag. Here, we assess the tolerance susceptibility of tissue-resident memory CD8 T cells in mice to these two forms of tolerogen. Soluble peptide specifically eliminated the majority of memory CD8 cells present in both lymphoid and extralymphoid tissues including lung and liver, but was unable to reduce the number present in the CNS. In contrast, systemic cross-presentation of Ag by dendritic cells resulted in successful elimination of memory cells only from the spleen, with no significant reduction in the numbers of tissue-resident memory cells in the lung. The fact that tissue-resident memory cells were unable to access cross-presented Ag suggests that either the memory cells in the lung do not freely circulate out of the tissue, or that they circulate through a region in the spleen devoid of cross-presented Ag. Thus, although tissue-resident memory cells are highly susceptible to tolerance induction, both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are successfully deleted from specific tissues.     

Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.     

Theoretical considerations of the role of antigen structure in B cell activation

Thymus-independent antigens generally are polymeric molecules with repeating arrays of antigenic determinants. Immunological studies of the activity of haptenated thymus-independent antigens have shown that small changes in hapten density can transform a polymeric antigen from nonimmunogenic to immunogenic, and from immunogenic to tolerogenic. In this paper we compute the equilibrium configuration of a linear flexible, haptenated polymer absorbed to a B cell surface, and correlate configurational features of the molecule with its immunological functioning. A polymeric molecule bound to a cell generally will not lie entirely on the surface; rather there will be sections that form loops extending into solution, separated by tightly bound sections, or trains. Trains link antibody receptors on the B cell surface in a fashion that restricts their mobility. Thus trains cause restrictive cross-linking. Our computations show that there is a critical hapten density below which the polymer does not bind to the surface. At hapten densities slightly above the critical density, the polymer binds weakly to the surface with a configuration dominated by a few, rather long loops. These loops cross-link receptors, but do so without bringing the cross-linked receptors into close proximity and without substantially restricting their motion. Long loops thus cause unrestrictive cross-linking. As the hapten density increases, the average loop length decreases and the average train length increases. Thus cross-linking becomes restrictive. In this density range, immune stimulation is observed. At high hapten densities long trains form, separated by few, very short loops and almost all receptors are cross-linked. Consequently cross-linking may be overly restrictive, freezing receptors into place and generating an abundance of cross-linking or other signals that induce a state of immunological tolerance.     

Interruption of tolerance toward a haptenic initiator of delayed-type hypersensitivity

We have found that an immunomodulator, dextran, prevents initiation of tolerance for an unrelated haptenic stimulus of contact hypersensitivity, picryl chloride. To prevent tolerance optimally, 1 to 10 mg of dextran per 25g mouse was required to be injected i.v. 2 hr before administering tolerogen. The immunomodulator was seen to be effective in mice with diverse major histocompatibility backgrounds. Furthermore, prevention of tolerance was seen to be independent of the condition of the hapten used to initiate unresponsiveness, since tolerance was abrogated when attempted with uncoupled hapten or with hapten attached to allogeneic or syngeneic spleen cells. In addition, administering dextran before tolerogen did not convert the tolerogenic signal into an immunogenic one so that hapten-specific DTH could be detected. While the precise mechanism of dextran's interruption of immunologic unresponsiveness for DTH has yet to be elucidated, the principle that tolerance can be readily and easily interrupted with this immunomodulating agent has been firmly established.