Expansion of neonatal tolerance to self in adult life: I. The role of a bacterial adjuvant in tolerance spread

Nir Grabie1, Ishay Wohl1, Sawsan Youssef1, Gizi Wildbaum1 and Nathan Karin1,2

1 Department of Immunology, Bruce Rappaport Faculty of Medicine and
2 Rappaport Family Institute for Research in the Medical Sciences, Technion, POB 9697, Haifa 31096, Israel

Correspondence to: N. Karin


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell neonatal tolerance to self evolves perturbation of the Th1/Th2 balance towards Th2-type self-specific T cells. In the current study we have demonstrated that a tolerant state could be extended to another encephalitogenic determinant only if the neonatally tolerizing determinant was co-administered in adult life with an emulsion of Mycobacterium tuberculosis (i.e. complete Freund's adjuvant). The mechanisms underlying tolerance elicitation and expansion were then explored by an in vitro system in which indirect suppression could be measured. Addition of a tolerizing epitope to splenic T cells from neonatally tolerized animals induced a marked suppression of the anti-MT response. This response could be restored by neutralizing antibodies to IL-4. In contrast, the neutralizing antibodies to IL-4 had no affect on the response of these cells to the tolerizing determinant. These findings are highly significant not only because they explore the important role of microbial antigens in neonatal tolerance, but also because they distinguish, for the first time, between tolerizing and tolerized T cells.

Keywords: complete Freund's adjuvant, determinant spreading, experimental autoimmune encephalomyelitis, Mycobacterium tuberculosis, myelin basic protein, neonatal tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The autoimmune response of T cells to components of the central nervous system (CNS) begins with recognition of a single or limited number of self-determinants and then expands into a reaction to several self determinants on the same molecule, termed intramolecular epitope spreading, or to other molecules within the nervous system, termed intermolecular epitope spreading (14). Thus, Lewis rats immunized with myelin basic protein (MBP) emulsified in complete Freund's adjuvant (CFA) to induce experimental autoimmune encephalomyelitis (EAE) first mount a primary T cell response to an encephalitogenic epitope encompassed by residues 68–86 (p68–86) of MBP and then to a secondary epitope consisting of residues 87–99 (p87–99) (1,2). These epitopes do not share cross-activating determinants. Yet, immunization of Lewis rats with one of these epitopes induces a response that spreads to the other one (4,5). This type of response involving epitope spreading has also been named a `trans-acting' or `cryptic' response (37).

The development of autoimmunity requires that T cells reactive to self-antigens escape elimination in the thymus. These self-reactive T cells are then activated in the periphery where they can provoke damage in specific organs (8,9). It has been long postulated that administration of an epitope in the embryonic or neonatal period may evoke its deletion of its recognition from the T cell repertoire of the adult. The observations that neonatal administration of one epitope initiated tolerance only to itself and not to other linked determinants of a tolerizing molecule (10) could be explained by the above hypothesis. After all, why should deletion of such determinant-specific T cells influence the T cell reactivity to a non-cross-reactive determinant? Indeed, neonatal administration of one MBP encephalitogenic determinant rendered resistance to disease induced only by that determinant and not by a linked epitope (5,6).

Based on their cytokine profile CD4+ T cells fall into at least three subfamilies, Th1 cells that produce large amounts of IFN-{gamma} and tumor necrosis factor (TNF)-{alpha}, but to a much lesser extent IL-4 or IL-10, and Th2 cells that that produce IL-4, IL-10, IL-13 and, to a much lesser extent, IFN-{gamma} and TNF-{alpha} (1120), and possibly Th3 cells that produce high amounts of transforming growth factor-ß (21). Alteration of the balance in favor of tolerizing T cell selection rather than across the board T cell elimination has recently been suggested to play a pivotal role in inducing and maintaining the tolerant state in antigen-specific T cell reactivity (2225). The current study uses the well-defined model of EAE to investigate key parameters in the expansion of a state of tolerance to different portions of the MBP molecule. The basic strategy has been to expose neonates to one MBP determinant and to determine the condition by which the above tolerant state could be extended to the other linked determinant. We now show that a tolerant state could be extended between linked determinants on MBP, only if the neonatally tolerizing determinant was co-administered in adult life with an emulsion of Mycobacterium tuberculosis (MT), i.e. CFA. Co-introduction with incomplete Freund's adjuvant (IFA) failed to induce tolerance. Thus, the study shows for the first time that microbes not only direct pathogenic T cell function, but are also involved in the initiation of the counter-restraining activity of tolerizing T cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rats
Pregnant Lewis rats, ~5 days before expected delivery, were purchased from Harlan (Jerusalem, Israel) and kept under pathogen-free conditions at our animal facility.

Peptide antigens
MBP p87–99, VHFFKNIVTPRTP, and MBP p68–86, YGSLPQKSQRSQDENPV, were synthesized on a MilliGen 9050 peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC. Structure was confirmed by amino acid analysis and mass spectroscopy. Only peptides that were >95% pure were used in our study.

Induction of neonatal tolerance
Tolerizing peptides at a concentration of 1 mg/ml were dissolved in PBS and emulsified with an equal volume of IFA (Difco, Detroit, MI). Within the first 24–72 h of birth newborn rats were immunized i.p. with 0.1 ml of the emulsion. Only female rats were later selected for experiments.

Active EAE induction
Active EAE was induced as described elsewhere (26). MBP p68–86 or MBP p87–99 peptides at a concentration of 1–1.5 mg/ml were dissolved in PBS and emulsified with an equal volume of IFA supplemented with 4 mg/ml heat-killed Mycobacterium tuberculosis (MT) H37Ra in oil (Difco, Detroit, MI). Rats were immunized s.c. in the hind foot pads with 0.1 ml of the emulsion and monitored daily for clinical signs by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, front and hind limb paralysis; 4, total body paralysis.

Culture media
Dulbecco's modified Eagle's medium (Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 2-mercaptoethanol (5x10–5 M), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 µ/ml), streptomycin (100 µg/ml) and 1% heat-inactivated normal Lewis rat serum was used as a stimulation medium.

Antigen-specific T cell proliferation assays
Lewis rats were immunized with various MBP peptides as described above. Nine to 10 days later draining lymph node (DLN) cells (DLNC) or spleen cells were suspended in stimulation medium and cultured in U-shape 96-well microculture plates (2x105 cells/well) for 72 h at 37°C in humidified air containing 7.5% CO2. For the trans acting antigen-specific proliferation assay spleen cells (2x105 cells/well) were plated together with MT (50 µg/ml) with, or without, additional MBP peptides at different concentrations. Each well was pulsed with 2 µCi of [3H]thymidine (sp. act. 10 Ci/mmol) for the final 18 h. The cultures were then harvested on fiberglass filters and the proliferative response expressed as c.p.m. ± SD or as stimulation index (SI) (mean c.p.m. of test cultures divided by mean c.p.m. of control cultures).

Cytokine determination
Spleen cells were stimulated in vitro (107 cells/ml) in 24-well plates (Nunc, Roskilde, Denmark) with 100 µM p68–86. After 48 h of stimulation, supernatants were assayed by semi-ELISA kits, that include antibody pairs and recombinant rat cytokines, as follows: IFN-{gamma}, rabbit anti-rat IFN-{gamma} polyclonal antibody (CY-048; Innogenetics, Zwijnaarde, Belgium) as a capture antibody, biotinylated mouse anti-rat mAb (CY-106 clone BD-1; Innogenetics) as a detection antibody and alkaline phosphatase-streptavidin (cat. no. 43-4322; Zymed, San Francisco, CA) with rat recombinant IFN-{gamma} as a standard (cat. no. 3281SA; Gibco/BRL, Gaithersburg, MD); IL-10, commercial semi-ELISA kit for the detection of rat IL-10 (PharMingen, San Diego, CA); IL-4, mouse anti-rat IL-4 mAb (24050D OX-81; PharMingen) as a capture antibody and rabbit anti-rat IL-4 biotin-conjugated polyclonal antibody (2411-2D; PharMingen) as second antibody. Recombinant rat IL-4 purchased from R & D Systems (Minneapolis, MN; 504-RL) was used as a standard. For each cytokine results of triplicates are presented.

Statistical analysis
Significance of differences was examined using Student's t-test. A value of P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The development and spreading of the T cell proliferative response in DLN and spleen of rats immunized with each of the above encephalitogenic epitopes in six independent consecutive experiments, all of which demonstrated the same pattern of results, were followed (Fig. 1Go). Rats immunized with the major determinant (p68–86) developed an early DNLC response to the immunizing epitope which peaked on day 7 (SI = 3.9), decreased within 2 days to SI = 2.33 (P < 0.01) and then to SI = 1.53 on day 11, which correlated with the onset of paralytic disease (Fig. 1E and AGo). Reduction in anti p68–86-specific T cell reactivity in DLNC (day 9) was followed by its reciprocal increase in the spleen (SI = 11.5). This response decreased at the onset of disease as well as day 11 (SI = 3.66). Spreading of anti-p68–86 reactivity from DLN to the spleen was followed by the development of a response to the subdominant epitope (p87–99) (a cryptic response) which peaked ~48 h after the response to the dominant epitope in both DLN and spleen. This response was significantly lower than the one obtained to the dominant epitope (Fig. 1E and CGo, P < 0.01).



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Fig. 1. Dynamics of determinant spreading in EAE. MBP p68–86 (YGSLPQKSQRSQDENPV) or MBP p87–99 (VHFFKNIVTPRTP) at a concentration of 2 mg/ml were dissolved in PBS, and emulsified with an equal volume of IFA supplemented with 4 mg/ml heat-killed MT H37Ra in oil and used for active induction of EAE. At 6 weeks of age female Lewis rats were immunized s.c. in the hind foot pads with 0.1 ml of either p68–86/CFA (A, C and E) or p87–99/CFA (B, D and F) and monitored daily for clinical signs by an observer blind to the treatment protocol (A and B). EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, front and hind limb paralysis; 4, total body paralysis. Every other day four rats were sacrificed. Splenic T cells (C and D) and DLN (E and F) were tested for their in vitro proliferative response to 100 mM of either p68–86 (black bars) or p87–99 (striped bars) exactly as we described in detail elsewhere (27,28). Results are shown as mean clinical score ± SE of six rats in each group (A and B) and as mean SI ± SE (quadruplicates) (C–F).

 
Rats immunized with the subdominant epitope of MBP exhibited a late DLN and spleen T cell response to the immunizing peptide, which peaked simultaneously with the development of a cryptic response to the dominant epitope. Onset of disease was delayed accordingly (Fig. 1B, D and FGo).

Each epitope was assessed for its ability to induce neonatal resistance (neonatal tolerance) to disease induced either by itself and or by the other epitope. Each tolerizing epitope, at a concentration of 1 mg/ml, was dissolved in PBS and emulsified with an equal volume of IFA. Within the first 24–72 h after birth newborn rats were immunized i.p. with 0.1 ml of the emulsion. Control neonates were immunized with the emulsion of IFA and PBS alone. Newborn rats neonatally tolerized by p68–86 acquired long-lasting resistance to EAE induced by p68–86/CFA (Fig. 2AGo) but not by p87–99/CFA (Fig. 2BGo) (one of six versus six of six, with mean maximum score 0.16 ± 0.1 versus 1.8 ± 0.2, P < 0.01). Rats neonatally tolerized by p87–99 acquired long-term resistance to EAE induced by p87–99/CFA (Fig. 2BGo) but not by p68–86/CFA (Fig. 2AGo) (one of six versus six of six, mean maximum score 0.16 ± 0.1 versus 2.5 ± 0.2, P < 0.01). Thus, each epitope could render resistance to EAE induced by itself but not to EAE induced by the other MBP peptide.



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Fig. 2. Determinant spreading in neonatally tolerized rats is dependent on subsequent co-immunization with the tolerizing determinant and MT. MBP p68–86 or MBP p87–99 at a concentration of 1 mg/ml were dissolved in PBS and emulsified with an equal volume of IFA. Within the first 24–72 h after birth newborn rats were immunized i.p. with 0.1 ml of p68–86/IFA (empty bars, empty circles), p87–99/IFA (gray bars, gray squares) or with IFA alone (black bars, black squares). Only female rats were later selected for experiments. At 6 weeks of age these rats (six per group) were subjected to active EAE induced by either p68–86/CFA (A, C, E and G) or by p87–99/CFA (B, D, F and H) as described in Fig. 1Go and monitored daily for clinical signs by an observer blind to the treatment protocol (A and B). At the peak of splenic T cell response (days 9 and 11 for rats with developing EAE induced by either p68–86 or p87–99 accordingly) splenic T cells from each group were monitored for their proliferative response against 100 µM of either p68–86 (C and D), p87–99 (E and F) or MT (G and H). Results are shown as mean clinical score ± SE (A and B) or as SI ± SE (C–H).

 
At the time when the peripheral T cell response to each immunizing epitope peaked in positive control rats (day 9 or 11 in active disease induced by either p68–86/CFA or p87–99/CFA respectively) splenic T cells from each group were tested for their proliferative response to the determinant with which disease was induced and to the other linked determinant. All EAE-resistant rats exhibited a profound reduction in antigen-specific T cell response to both the tolerizing epitope and also to the linked determinant (Fig. 2CGo, SI = 0.9 ± 0.2 versus SI = 11.4 ± 0.4. in control rats, P < 0.001, and cryptic response to p87–99 in the same rats, Fig. 2EGo, SI = 1.23 ± 0.2 versus SI = 3.43 ± 0.4., P < 0.001; response to p87–99 in control rats compared with that in p87–99 tolerized rats, both immunized with p87–99/CFA in adult life, Fig. 2FGo, SI = 5.34 ± 0.3 versus SI = 1.38 ± 0.2, P < 0.001, and cryptic response to p68–86 in the same rats, Fig. 2DGo, SI = 5.1 ± 0.4 versus SI = 1.4 ± 0.3, P < 0.001). There was no reduction in splenic T cell proliferation to the MT present in the immunizing emulsion in any of the above EAE-resistant groups (Fig. 2G and HGo). Rats that were neonatally tolerized by one MBP epitope and then immunized in adult life with the other epitope emulsified in CFA all developed EAE (Fig. 2A and 2BGoGo) and exhibited no reduction in their peripheral T cell response to the dominant nor to the subdominant epitope of MBP (Fig. 2C-c and 2F-bGo). Thus induction of neonatal tolerance is determinant specific. Yet, once an animal reaches a tolerant state the primary T cell response to both linked determinants, but not to MT, is markedly suppressed.

To determine whether a subsequent immunization of a tolerizing determinant may provide the means for spreading of the tolerant state between linked determinants at the target organ, Lewis rats were neonatally tolerized with one encephalitogenic determinant and immunized at adult life with the other linked determinant emulsified in CFA, with or without a simultaneous challenge of the tolerizing determinant in the opposite leg. All rats that were neonatally tolerized with p87–99/IFA were susceptible to EAE induced by p68–86/CFA and developed severe disease that persisted for 5–7 days (six of six rats per group), but were markedly resistant to EAE induced by p68–86/CFA plus p87–99/CFA simultaneously injected in opposite legs (Fig. 3CGo, one of six compared with six of six, P < 0.01). Resistance was associated with a marked reduction in anti-p87–99 as well as anti-p68–86 splenic T cell proliferative responses (in response to p87–99, SI = 1.5 ± 0.37 in p87–99 tolerized rats and 1.2 ± 0.34 in p68–86 tolerized rat versus SI = 4.5 ± 0.35 in control rats, P < 0.001; in response to p68–86, SI = 1.3 ± 0.72 in p87–99 tolerized rats and 1.3 ± 0.37 in p68–86 tolerized rat versus SI = 5.2 ± 0.35 in control rats, P < 0.001), but not to the MT with which the tolerizing epitope was given at adult life (SI = 12.4 ± 0.87 in p87–99 tolerized rats, SI = 10.1 ± 0.87 in p68–86 tolerized rats and SI = 9.2 ± 1.36 in control rats).



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Fig. 3. Determinant specificity in neonatal tolerance induced by linked MBP determinants. MBP p68–86, MBP p87–99 or ovalbumin (grade VII; Sigma) at a concentration of 1 mg/ml were dissolved in PBS and emulsified with an equal volume of IFA. Within the first 24–72 h after birth, newborn Lewis rats were immunized i.p. with 0.1 ml of p68–86/IFA (empty circles), p87–99/IFA (gray squares), ovalbumin/IFA (empty diamonds) or with IFA alone (black squares). Only female rats were later selected for experiments. At 6 weeks of age these rats (six per group) were subjected to active EAE induced by p68–86/CFA in one leg + CFA alone in the opposite leg (A), p87–99/CFA in one leg + CFA alone in the opposite leg (B), p68–86/CFA in one leg + p87–99/CFA in the opposite leg (C), p68–86/CFA in one leg + p87–99/IFA in the opposite leg (D) or p68–86/CFA in one leg + ovalbumin/CFA in the opposite leg (E). All rats were monitored daily for clinical signs by an observer blind to the treatment protocol. Results are shown as mean clinical score ± SE.

 
The experiment described above was repeated with the exception that the challenging antigen was emulsified in IFA rather than CFA. Thus, Lewis rats were neonatally tolerized with one encephalitogenic determinant and immunized at adult life with the other linked determinant emulsified in CFA (to induce active EAE), with a simultaneous challenge of the tolerizing determinant, emulsified in IFA, in the opposite leg. These neonatally tolerized rats did not acquire any resistance to EAE (Fig. 3DGo, incidence six of six) suggesting, for the first time, that in vivo activation of the antigen-specific tolerizing cells may be dependent on the use of MT as a part of an adjuvant.

Rats treated neonatally with ovalbumin/IFA and challenged with p68–86/CFA plus ovalbumin/CFA, simultaneously injected in opposite legs, did not demonstrate any EAE resistance (Fig. 3EGo). Ovalbumin failed to induce trans-acting neonatal tolerance either because it was not continually accessible to the immune system or because it was not displayed in proximity to the encephalitogenic determinant at the site of inflammation (i.e. the brain).

In another experiment, rats were neonatally tolerized by p87–99/IFA and subsequently challenged in adult life with p68–86/CFA plus either p87–99/CFA or p87–99/IFA, simultaneously injected in opposite legs. The splenic T cell proliferative response against p68–86 was markedly reduced in rats that were challenged in adult life with p68–86/CFA plus p87–99/CFA and not in those challenged with p68–86/CFA plus p87–99/IFA (Fig. 4, 5GoGo.1 ± 0.4 versus 1.3 ± 0.2, P < 0.001), indicating that MT-dependent disease inhibition is associated with T cell suppression.



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Fig. 4. A subsequent immunization of neonatally tolerized rats with a linked determinant in CFA induces a marked reduction in T cell response to both determinants. MBP p87–99 at a concentration of 1 mg/ml was dissolved in PBS and emulsified with an equal volume of IFA. Within the first 24–72 h after birth newborn rats were immunized i.p. with 0.1 ml of the above emulsion or with IFA alone. At 6 weeks of age these rats (six per group) were challenged with p68–86/CFA in one leg + CFA alone in the opposite leg, p68–86/CFA in one leg + p87–99/CFA in the opposite leg or with p68–86/CFA in one leg + p87–99/IFA in the opposite leg. Nine days later splenic T cells from all rats in each of the above groups were tested for their proliferative response against p68–86. Results are shown as SI ± SE.

 


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Fig. 5. IL-4 neutralizing antibodies reinstate trans-acting suppression in neonatally tolerized rats. MBP p68–86 at a concentration of 1 mg/ml was dissolved in PBS and emulsified with an equal volume of IFA. Within the first 24–72 h after birth newborn rats were immunized i.p. with 0.1 ml of the above emulsion (A) or with IFA alone (B). At 6 weeks of age these rats (four per group) were challenged with p68–86/CFA. Nine days later splenic T cells were cultured together with either p68–86 (100 µg/ml), MT (100 µg/ml) or their combination, with or without the addition of anti IL-4 neutralizing antibodies (rabbit anti-rat IL-4 polyclonal antibody 2411-2D) at a final concentration of 150 ng/ml. Results of the proliferative response of each group are shown as SI ± SE.

 
Alteration of the balance in favor of Th2 cell selection rather than generalized T cell elimination has been suggested to play a pivotal role in inducing and maintaining neonatal tolerance (2225). We have evaluated this possibility. Spleen cells from rats tolerized neonatally with p68–86/IFA were obtained 9 days after adult challenge with the tolerizing epitope and cultured for 48 h in stimulation medium. A profound increase in IL-4 production was observed in spleen cells from tolerized rats (255 ± 30 versus 2.5 ± 1 pg/ml, P < 0.0001). The above increase was accompanied by a significant reduction in IFN-{gamma} production (<1000 versus 6100 ± 400 pg/ml, P < 0.0001). To further investigate the possible role of IL-4 in neonatal tolerance, an in vitro system, in which indirect (trans-acting) suppression could be measured, has been established. Addition of the tolerizing epitope to splenic T cells from tolerized animals induced a marked trans-acting suppression of their anti-MT response (Fig. 5AGo, 12 ± 1.2 versus 2.5 ± 0.8, P < 0.0001). This response could be restored by anti-IL-4 neutralizing antibodies (Fig. 5AGo, 11.6 ± 1.3 versus 10.7 ± 1.1). In contrast, neither anti-IL-4 antibodies (Fig. 5A and BGo, 1.5 ± 0.4 and 2.1 ± 0.2 versus 6.4 ± 0.4 and 6.3 ± 0.6, P < 0.001) nor the combination of anti-IL-4 and anti-IL-10 antibodies (data not shown) could restore the direct response of these cells to the tolerizing epitope. Addition of the tolerizing epitope to splenic T cells from control EAE animals did not induce trans-acting suppression of their anti-MT response (Fig. 5BGo, 9.5.2 ± 1.3 versus 9.1 ± 1.1). Our data, therefore, suggest that based on the ability of anti-IL-4 neutralizing antibodies to reinstate T cell proliferation, tolerizing and tolerized T cells are distinguishable.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously used molecular biologic techniques to demonstrate that T cell homing to the site of inflammation is a multi-sequential event that includes a selective stage in which antigen-specific T cells interact with their target antigen resulting in activation of the blood–brain barrier to allow the accumulation of a non selective influx of endogenous T cells into the CNS which correlates with disease onset (2730). It is, therefore, likely that epitope spreading entails non-selective entrance of non-activated circulating T cells into the CNS during the inflammatory breach in the blood–brain barrier and their activation, possibly for the first time, at the site of inflammation. The above cryptic response is detectable in T cells isolated directly from the CNS (4,31) as well as in T cells isolated at the same time from the periphery (4,5,31). It is therefore plausible that following their first in vivo activation at the target organ some of these in vivo activated T cells circulate back to the periphery (32) where they can be easily isolated. This system has allowed us to re-evaluate the mechanism underlying T cell homing, selection and activation in a target organ in neonatally tolerized animals.

In the present study determinant spreading in EAE has led to the elucidation of new concepts underlying neonatal tolerance. We now show that not only activation of pathogenic T cells to non-cross activating determinants may occur, termed epitope spreading, but also that the tolerant state in T cells exhibits similar determinant spreading. Such a mechanism has been suggested for oral tolerance (33). Moreover, the current study uses the well-defined system of EAE to demonstrate, for the first time, the role of microbes in the spreading and maintenance of the tolerant state to self determinants.

It has been argued that a co-stimulatory signal is required for the initiation of adaptive immune response (34). From an evolutionary point of view this may reflect the history of host defense. The immune system's primary driving force is the need to detect and protect against danger (35). Efficient performance of this role requires positive and negative communication with an extended network (35). So far various studies have demonstrated the contribution of viral and microbial organisms to the initiation of direct and trans-acting autoimmune encephalomyelitis, and possibly multiple sclerosis (3639). Our study shows for the first time that microbes not only direct pathogenic T cell function, but are also involved in the initiation of the counter-restraining activity of tolerizing T cells. It is apparent that macrophages alone are not an immediate target of the MT components, otherwise newborn rats exposed to one MBP determinant and challenged in adult life with the other determinant in CFA together with the tolerizing determinant in IFA should show EAE resistance. We think that either the tolerizing T cells are the immediate target of MT components or that MT components interfere in co-signaling between antigen-presenting cells and the tolerizing T cells.

It has recently been suggested that neonatal T cell tolerance to self arises from a perturbation in their immune reactivity, rather than from a general elimination of normally occurring autoreactive T cells (2225). Neonatal administration of an MBP determinant protects against EAE induced by subsequent administration of this determinant in CFA (5,6).

With the exception of a single recent study carried out in genetically modified mice (40), most investigators agree that Th1 cells selected in response to various auto-antigens transfer T cell-mediated autoimmune diseases, whereas IL-4 secreting Th2 cells, selected in response to these same antigens, either inhibit or exert no profound effect on the inflammatory process (15,4153). It is thus conceivable that a shift in the Th1/Th2 balance towards determinant-specific Th2 cells in neonatally tolerized animals could be protective because it leads to a substantial quantitative reduction in the amount of antigen specific pro-inflammatory Th1 cells. While still to be proven, it is also possible that a subpopulation of T cells, selected by neonatal administration of tolerizing antigen and subsequent challenge with bacterial adjuvant at adult life, display a regulatory effect on the function of antigen-specific pro-inflammatory T cells. This may well explain spreading of the tolerant state between MBP-linked determinants in EAE (Fig. 2Go) as well as between various antigenic determinants associated with the development of diabetes in NOD mice (54) or between acetylcholine receptor {alpha} subunit determinants in myasthenia gravis (55). The current study, which shows, for the first time, that a response to MT can be suppressed in p68–86 tolerized mice by the addition of the tolerizing determinant, strongly supports this hypothesis (Fig. 5Go). Moreover, we show here for the first time that IL-4 neutralizing antibodies may reinstate the above trans-acting suppression, but not the direct response to the tolerizing determinant (Fig. 5Go). Thus, based on the ability to reinstate an in vitro proliferative response, our data distinguishes between tolerized T cells, to which pro-inflammatory Th1 cells probably belong, and tolerizing T cells. These tolerizing T cells do produce high levels of IL-4 and low levels of IFN-{gamma} as typical Th2 cells do; hence, their in vivo potentiation requires the subsequent immunization of their target antigen together with CFA and even then they mount a poor in vitro response to their target determinant.

Self-reactive T cells, including tolerizing T cells may, potentially, be subjected to a continuing exposure to their target antigen. We have recently determined minimal requirements for peripheral tolerance induction and showed that an engagement of one TCR-binding site and one MHC anchor to an antigenic determinant (p87–99) is sufficient for induction of determinant-specific T cell tolerance, whereas engagement of four different amino acids to MHC and three other amino acids to the TCR are essential to mount a substantial in vitro proliferative response in these T cells (56). These data suggest that the immune system has been selected to prefer restraining, rather than activating, self-reactive effector T cells. Perhaps the incompetence of neonatally tolerizing T cells to mount a substantial proliferative response against their target self determinants is a mechanism by which their powerful suppressive competence is restrained as well.


    Acknowledgments
 
We would like to thank Dr H. Gershon for creative discussions and for reading the manuscript. This study was supported by Israel Cancer Research Foundation (ICRF), Israel Science Foundation, Israel Ministry of Science and Arts, Israel Ministry of Health, and the Technion VPR–Albert Goodstein Fund.


    Abbreviations
 
CFAcomplete Freund's adjuvant
CNScentral nervous system
DLNdraining lymph node
DLNCdraining lymph node cell
EAEexperimental autoimmune encephalomyelitis
IFAincomplete Freund's adjuvant
MBPmyelin basic protein
SIstimulation index
TNFtumor necrosis factor

    Notes
 
Transmitting editor: L. Steinman

Received 12 January 1999, accepted 17 February 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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