The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Illés, Z.
Right arrow Articles by Kuchroo, V. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Illés, Z.
Right arrow Articles by Kuchroo, V. K.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2005, 175: 1491-1497.
Copyright © 2005 by The American Association of Immunologists

T Cell Tolerance Induced by Cross-Reactive TCR Ligands Can Be Broken by Superagonist Resulting in Anti-Inflammatory T Cell Cytokine Production1

Zsolt Illés, Hanspeter Waldner2, Jayagopala Reddy, Estelle Bettelli, Lindsay B. Nicholson3 and Vijay K. Kuchroo4

Center for Neurologic Diseases, Harvard Institute of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cross-reactive activation of potentially autoreactive T cells by high-affinity nonself ligands may be important in breaking self-tolerance in autoimmunity. In a mouse transgenic for a cross-reactive TCR, we have previously shown that a hyperstimulating altered peptide ligand, L144, induced unresponsiveness to the self peptide, proteolipid protein 139–151. In this study, we demonstrate that a superagonist ligand can break T cell tolerance induced by the lower affinity cognate Ag. T cells tolerant to the cognate ligand, Q144, responded to superagonist, L144, by proliferation and the production of mainly IL-4 and IL-10 in vitro. In contrast, T cells that were tolerized to the superagonist were unable to respond to any peptide that cross-reacted with the transgenic TCR. Low-dose immunization with the superagonist L144 was able to break tolerance to the cognate ligand in vivo and resulted in a blunted proliferative response with production of Th2 cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Studies of the maintenance of self-tolerance and the mechanisms by which this tolerance can be broken are essential to an understanding of the pathogenesis of autoimmune diseases. Central tolerance plays an important role deleting self-reactive T cells in the thymus (1, 2, 3). Yet autoreactive T cells do exist in the peripheral repertoire. This may be due to either incomplete negative selection of T cells in the thymus (1, 2, 3, 4) or to the inherently cross-reactive nature of TCRs (5, 6). Several mechanisms of peripheral tolerance that prevent the activation of these autoreactive T cells have been proposed. In addition to the role of regulatory/suppressor T cell populations, ignorance of self-Ags (7, 8), down-regulation of the TCR or coreceptors (9, 10, 11), and the complete elimination of autoreactive T cell clones from the peripheral repertoire (12, 13, 14) can result in tolerance against self. Functional unresponsiveness of T cells, i.e., anergy, is another potential mechanism to silence self-reactive T cells. Although anergy was originally defined as a defect in TCR-dependent proliferation and production of IL-2 in vitro (15, 16, 17), this form of T cell unresponsiveness has been demonstrated in several in vivo models as well (18, 19, 20, 21, 22, 23, 24, 25, 26).

In the face of activation these mechanisms of peripheral tolerance can fail, and cross-reactive foreign Ag can induce a detrimental T cell response that leads to autoimmune disease (27, 28). Because T cells expressing a high-affinity TCR to self-Ags are deleted in the thymus by negative selection, cross-reactive foreign Ags can activate low-affinity self-reactive T cells; however, the conditions and mechanisms by which cross-reactive non-self-Ags break self-tolerance and induce autoimmunity have not been elucidated. Memory T cells have a lower threshold for activation; thus, when activated during an immune response against high-affinity foreign Ags, cross-reactive low-affinity self-ligands may potentially reactivate an autoreactive T cell response. We have recently proposed a mechanism which can counteract this cross-reactive memory T cell response. Using a TCR transgenic (tg)5 mouse model, we showed that stimulation of T cells with a superagonist ligand, L144, tuned the T cells and this resulted in nonresponsiveness rather than hyperresponsiveness to the self-ligand (29). This mechanism may contribute to the maintenance of peripheral tolerance, where self-peptides fail to induce deletion, possibly due to their low affinity for the TCR or to the poor expression of self-Ag in the thymus.

In contrast, in autoreactive T cells that have been tolerized by self-ligands, cross-reactive superagonists may break the tolerant state, resulting in a potentially detrimental autoimmune response. To further understand how peripheral tolerance is maintained against cross-reactive superagonists, we investigated here whether a superagonist peptide can break tolerance induced by high-dose soluble cognate ligand.

To examine the role of cross-reactive superagonist in induction and breaking of peripheral tolerance, we used 1B6 TCR tg mice (29). These mice express a transgenic TCR from a T cell clone derived from a mouse immunized with the altered peptide (Q144; HSLGKQLGHPDKF), in which glutamine (Q) has replaced the primary TCR contact tryptophan (W) in the autoantigen PLP 139–151 (HSLGKWLGHPDKF). The 1B6 TCR is cross-reactive with a number of ligands beside the cognate ligand Q144 in a hierarchical fashion. The self autoantigenic peptide PLP 139–151 (W144) is a weak agonist, whereas Y144 stimulates tg cells more strongly than Q144 and the superagonist L144 is a hyperstimulatory ligand (30).

Using this system, we show here that superagonist L144 can break tolerance to Q144. Although the response is blunted, the cytokine profile was skewed to the anti-inflammatory cytokines IL-4 and IL-10, which may be critical for preventing development of autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female SJL mice, 6–8 wk old, were purchased from The Jackson Laboratory and housed under specific pathogen-free conditions at the Harvard Institutes of Medicine (Boston, MA). 1B6 TCR transgenic mice were generated in our laboratory as described previously (29). All of the mice were maintained in accordance with guidelines of the Committee on Animals of Harvard Medical School. Female mice, 8–12 wk old, were used in all the experiments. RAG-2-deficient mice were backcrossed onto the SJL/J background in our laboratory and 1B6 mice were further crossed onto the RAG-2-deficient SJL/J background.

Antigens

Peptide Ags were synthetized by F-moc chemistry and they were >90% pure. The following peptides were used in this study: proteolipid protein (PLP) 139–151 (W144) (HSLGKWLGHPDKF), Q144 (HSLGKQLGHPDKF), Y144 (HSLGKYLGHPDKF), and L144 (HSLGKLLGHPDKF). The relative binding of the PLP analog peptides to I-As compared with W144 is 0.6 (Q144), 0.6 (Y144), and 0.8 (L144) (29).

Antibodies

Transgenic 1B6 cells were stained by flow cytometry using a clonotypic biotinylated Ab, 1.16, as previously described (29). Streptavidin-PE or streptavidin-allophycocyanin was used as a secondary Ab (BD Pharmingen). CD4-allophycocyanin and CD4-FITC were purchased from BD Pharmingen. All of the staining reactions were conducted in 1x PBS containing 2% FCS (Sigma-Aldrich) and acquired using a FACSort flow cytometer (BD Biosciences) and analyzed by FlowJo software (Treestar; Amersham).

CFSE labeling and adoptive transfers

Lymph node cells (LNC) were isolated from tg 1B6 mice and CD3+ cells were enriched by negative selection using mouse T cell enrichment columns (R&D Systems). Ten million T cells were incubated with 1.5 µM CFSE in PBS for 5 min at room temperature. The labeled cells were washed once with ice-cold 100% FBS (Sigma-Aldrich) and washed twice with PBS. Five million cells were subsequently transferred i.v. into naive SJL wild-type (wt) mice. For some experiments, unlabeled T cells were transferred to mice.

Induction of anergy and immunizations

Recipient SJL mice were each injected i.p. with high-dose soluble peptides W144 (300 µg), Q144 (300 µg), Y144 (300 µg), or L144 (1, 10, 50, or 100 µg) in sterile PBS 2 days after transfer of 1B6 T cells. LNC were isolated 10 days after tolerization and analyzed by flow cytometry. Mice were immunized s.c. with 100 µg of Q144, 100 µg of Y144, or 1 or 10 µg of L144 in CFA 10 days after tolerization. LNC were isolated and analyzed 7 days after immunization.

Proliferation assays

LNC (2 x 105) were stimulated with the indicated concentrations of peptide or PMA plus ionomycin for 2 days. Cultures were pulsed with 1 µCi of [3H]thymidine during the last 16 h before cells were harvested. The incorporation of [3H]thymidine was determined using a Wallac scintillation counter. Supernatants from the above cultures were collected at 48 h, and cytokine levels were determined by ELISA according to the manufacturer’s instructions (BD Biosciences).

Cytokine measurement by ELISA

Supernatants obtained from cultures were collected after 48 h of activation and stored at –70°C until use. Secretion of IL-2, IL-4, IL-10, and IFN-{gamma} was measured by ELISA. Briefly, cytokine mAbs for IL-2, IL-4, IL-10, and IFN-{gamma} were coated to 96-well plates at a concentration of 1 µg/ml overnight at 4°C. The plates were washed and treated with blocking solution (Kirkegaard & Perry), followed by incubation of culture supernatants overnight at 4°C. The plates were subsequently washed and incubated with their corresponding biotinylated cytokine-detecting mAb (1 µg/ml) for 2 h (IL-2: JES6-A112 and JES6-5H4; IL-4: 11B11 and BVD6-24G2; IL-10: JES5-16E3 and SXC-1; IFN-{gamma}: R4-6A2 and XMG1.2). The plates were developed after adding avidin peroxidase and its substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Stimulation with superagonist L144 induces proliferation and alters cytokine production of tg T cells anergized with agonist ligands

To examine the effect of altered peptides on tolerized T cells, an in vivo high-dose tolerance system was set up in which 1B6 tg T cells were transferred into wild-type SJL/J mice (29, 30). The 1B6 tg mice express a rearranged transgenic TCR which responds to the altered peptide of PLP 139–151, Q144, in which the tryptophan (W) at 144 was replaced with glutamine (Q) (31). Besides the cognate ligand Q144, the tg 1B6 cells respond to different ligands in a hierarchical fashion: the PLP 139–151 peptide (W144) induces a weak proliferative response, whereas agonist Y144 stimulates cells at 1 log less concentration than Q144, and superagonist L144 is able to induce a proliferative response at a 105 times lower concentration than the cognate ligand Q144 (29, 30).

First, we examined whether transferred 1B6 tg T cells retain the potential to respond to different ligands in a hierarchical fashion. Purified T cells from lymph nodes of 1B6 transgenic mice were transferred into naive SJL/J mice. LNC of the recipients were isolated 10 days later, and the proliferative response to different ligands was examined and compared with LNC of naive 1B6 mice. LNC of the recipients were able to proliferate to all four ligands in a hierarchical fashion similar to naive 1B6 tg LNC (Fig. 1A). In contrast, LNC of naive SJL/J mice have an endogenous response to W144 (PLP 139–151) (1) and do not respond at all to Q144, Y144, or L144 (data not shown).



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 1. Proliferative response and cytokine production of 1B6 tg T cells following tolerization with high-dose soluble cognate peptide Q144. A, LNC (2 x 105) were isolated from wild-type SJL/J mice 10 days after transfer of 5 x 106 purified 1B6 T cells (recipient). LNC were cultured with PLP 139–151 (W144), Q144, Y144, or L144 at the indicated concentrations for 72 h. The proliferative response is shown as {Delta}cpm. B, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of soluble Q144 i.p. 2 days later, and proliferation of 2 x 105 LNC was examined in response to W144, Q144, Y144, and L144 after 10 days. Data represent mean {Delta}cpm ± SEM of five mice per group. C, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of soluble W144, Q144, Y144, or 10 µg L144 i.p. 2 days later. Percentages of 1B6 tg cells were determined in the lymph node 10 days after induction of tolerance by flow cytometry using a clonotypic mAb (1.16). D, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of Q144 i.p. 2 days later. Cytokine production was measured by cytokine ELISAs 10 days later in supernatants of LNC cultures activated with 0.01 ({blacksquare}) or 1 µg/ml ({square}) L144 for 48 h.

 
Next, we examined whether transferred 1B6 tg T cells can be tolerized by high dose soluble Ag. 1B6 tg T cells were transferred into naive SJL/J mice, and 2 days later, mice were injected with 300 µg of soluble Q144 peptide i.p. Ten days after tolerization, LNC of the recipients were isolated and pooled and the proliferative response to the tolerogenic cognate ligand Q144 was determined. As shown in Fig. 1B, the proliferative response to Q144 was greatly reduced in comparison to nontolerized recipients. This decreased proliferative response to Q144 was not due to clonal deletion, because the percentage of tg cells characterized by the double staining with CD4 and a clonotypic mAb 1.16 was similar in tolerized and untolerized mice (Fig. 1C).

We then examined whether tg T cells, tolerized with the cognate ligand Q144, were able to proliferate in response to weak agonist PLP 139–151 (W144), agonist Y144, and superagonist L144 peptides. As shown in Fig. 1B, LNC from mice tolerized with Q144 did not proliferate when stimulated with self-peptide W144 in vitro. We concluded that altered peptides, which induce weaker responses than the tolerogenic ligand, could not elicit the proliferation of tolerized T cells. This conclusion was further supported by the finding that if tolerance was induced by Y144, LNC of the recipients were tolerant to all of the ligands weaker than Y144, which included W144, Q144, and Y144 (data not shown). In contrast, superagonist L144 was able to induce proliferation when tg T cells were tolerized with cognate ligand Q144 (Fig. 1B) or agonist Y144 (data not shown). However, this proliferative response was blunted compared with LNC obtained from nontolerized recipients (Fig. 1, A and B). Moreover, the tolerant tg LNC did not produce IL-2, IFN-{gamma}, IL-4, and IL-10 in response to the tolerogenic ligand (data not shown), but they retained the capacity to secrete IL-2 and IFN-{gamma} after L144 stimulation (Fig. 1D). Remarkably, T cells tolerized with Q144 also produced IL-4 in response to L144 stimulation, although no IL-10 was detected. Production of IL-4 was not detected in nontolerized tg T cells, which produced only IL-2 and IFN-{gamma} upon activation with superagonist (Fig. 1D). Taken together, the data suggest that tolerized 1B6 T cells were unable to proliferate in response to the tolerogenic and low-affinity ligands, but they responded to superagonist L144, indicating a hierarchy in anergy. However, the response to L144 was altered compared with that of nontolerized cells in that proliferation to L144 was accompanied by IL-4 secretion with a concomitant decrease in IFN-{gamma} production.

Immunization with superagonist L144 results in expansion of Q144-tolerized T cells in vivo

After confirming that, after tolerization with the cognate ligand Q144, 1B6 tg T cells could still respond to superagonist L144 in vitro, we sought to examine whether exposure to L144 in an inflammatory environment can also break tolerance in vivo. Purified T cells obtained from RAG2-deficient 1B6 transgenic mice were transferred into SJL/J mice. Recipients were tolerized with 300 µg of Q144 2 days later and immunized with either 100 µg of Q144 or 1 µg of superagonist L144 10 days after the induction of tolerance. LNC were isolated 5 days later, and the percentage of 1.16+CD4+ T cells was determined. Tolerization with Q144 and combined tolerization/immunization with Q144 did not increase the percentage of 1B6 T cells (Fig. 2A). In contrast, in mice tolerized with Q144 and immunized with superagonist L144, the percentage of 1B6 tg T cells increased 3-fold, suggesting that immunization induced expansion of tolerized T cells in vivo (Fig. 2A). This was further confirmed by studying the expansion of cells in vivo using CFSE. CD3+ cells from 1B6 tg/RAG2-deficient mice were enriched, labeled with CFSE, and transferred into SJL/J mice. Recipient mice were tolerized with 300 µg of Q144 2 days later and immunized with either 100 µg of Q144 or 1 µg of L144 in CFA 10 days later. LNC were isolated 5 days after immunization, and CFSE staining of CD4+1.16+ T cells was examined by flow cytometry. A very low-level division of transferred 1B6 T cells was observed following tolerance induction with high-dose Q144 (data not shown). Immunization with 100 µg of Q144 in CFA also failed to induce proliferation of tolerized T cells. In contrast, immunization with low dose (1 µg) of L144 in CFA induced expansion of tolerized tg T cells (Fig. 2B). These data suggest that L144 not only elicited a response in Q144-tolerized T cells in vitro, but was also able to induce their proliferation in vivo.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 2. Expansion of Q144-tolerized 1B6 tg T cells following immunization with superagonist L144. A, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of soluble Q144 i.p. 2 days later (Qtol) and subsequently immunized with 100 µg of Q144 (Qtol/Qimm) or 1 µg of L144 in CFA (Qtol/Limm). The percentage of 1B6 tg cells was determined in the lymph nodes 5 days after immunization by flow cytometry using a clonotypic mAb (1.16). B, CFSE-labeled purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of soluble Q144 i.p. 2 days later and subsequently immunized with 100 µg of Q144 (Q144/CFA) or 1 µg of L144 in CFA (L144/CFA). Cell division was examined in gated 1.16+CD4+ cells 5 days later by flow cytometry.

 
Immunization with superagonist L144 breaks tolerance, but the response is blunted to the tolerogenic peptide in vitro

The in vivo expansion of Q144-tolerized T cells upon immunization with L144 could be due to two reasons: the tg T cells may have responded to the superagonist L144 as evaluated by assessing division in vivo, but still retained unresponsiveness to the tolerogen Q144 ligand. Alternatively, immunization with L144 might in fact break tolerance such that tg T cells lose unresponsiveness to the tolerizing peptide as well. To examine whether immunization with L144 could result in a loss of tolerance in Q144-tolerized 1B6 transgenic T cells, we determined their Q144-specific responses in vitro.

Five million enriched CD3+ 1B6 T cells were transferred to SJL/J mice. Recipient mice were tolerized with 300 µg of Q144 2 days later. Ten days after induction of tolerance, mice were immunized with either 100 µg of Q144 or 10 µg of L144 in CFA. Responses of LNC to Q144 were examined 5 days after immunization in three different experimental systems. We first determined the frequency of 1B6 tg T cells. In cultures stimulated with Q144, the percentage of Q144-tolerized T cells decreased, while immunization with superagonist, L144, overcame tolerance to Q144 and resulted in a 10-fold increase of 1B6 tg T cells in response to Q144 (Fig. 3A). In addition, LNC of mice tolerized with Q144 and subsequently immunized with L144 were able to proliferate in response to Q144 stimulation. This Q144-specific proliferation was however significantly lower than the response seen in T cells of nontolerized mice immunized with L144 or Q144 (Fig. 3B). This finding indicated that the immunization with superagonist L144 was able to break tolerance to Q144, but the response to Q144 was blunted compared with the response obtained from nontolerized, L144-immunized recipients. Similar data were obtained when mice were tolerized with Y144 followed with immunization with L144 (data not shown). Finally, the increased percentage of 1B6+CD4+ T cells in vitro suggested that the blunted proliferation was the response of 1B6 tg T cells and not of endogenously primed SJL/J T cells (Fig. 3A). To verify this, LNC of Q144-tolerized, L144-immunized recipients were labeled with CFSE and stimulated with 100 µg/ml Q144 in vitro for 3 days. We observed that 1.16+CD4+ tg T cells divided in response to Q144, indicating that immunization with L144 was indeed able to break the unresponsiveness of Q144-tolerized 1B6 tg T cells, although this response was reduced compared with untolerized recipients (Fig. 3C).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of immunization on the proliferation of tolerized 1B6 T cells in vitro. A, Purified 5 x 106 transgenic 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of soluble Q144 i.p. 2 days later and subsequently immunized with 10 µg of L144 in CFA (Qtol/Limm) or left unimmunized (Qtol). LNC were isolated 10 days later and stimulated with 100 µg/ml Q144 or medium alone in vitro. The percentage of 1B6 tg cells were determined 3 days later by flow cytometry using a clonotypic mAb (1.16). B, Purified 5 x 106 transgenic 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 300 µg of Q144 in PBS i.p. 2 days later (Qtol). After 10 days, mice were immunized with either 100 µg of the tolerogen Q144 (Qtol/Qimm) or 10 µg of L144 in CFA (Qtol/Limm). The proliferative response to the tolerogen Q144 was determined 5 days later and was compared with proliferation of cells from recipients, which were not tolerized with Q144 but were immunized with 100 µg of Q144 (Qimm) or 10 µg of L144 (Limm). C, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were left untreated or injected with 300 µg of Q144 in PBS i.p. 2 days later. After 10 days, mice were immunized with 10 µg of L144 in CFA or left unimmunized. LNC were isolated after 5 days, labeled with CFSE, and restimulated with tolerogen Q144 (100 µg/ml). Cell divisions were assessed in 1.16+CD4+ gated cells by flow cytometry. Open histograms indicate cell divisions of tg T cells stimulated with Q144, whereas filled histograms indicate unstimulated cultured tg T cells.

 
Superagonist breaks T cell tolerance but alters cytokine response

We examined the cytokine response to Q144 stimulation in immunized and tolerized mice. Immunization with Q144 or L144 without tolerization induced a strong IL-2 and IFN-{gamma} production upon stimulation with Q144 in vitro. In contrast, in LNC cultures of mice tolerized with soluble Q144, the production of IL-2 and IFN-{gamma} was significantly reduced. Remarkably, immunization of Q144-tolerized mice with L144 induced IL-4 and IL-10 production in LNC. In contrast, LNC from mice that were only tolerized with Q144 did not produce these cytokines (Fig. 4). Because immunization of mice with L144 did not induce IL-4 and IL-10 either, these cytokines were unlikely to be produced by endogenous non-tg T cells. These data indicated that immunization with superagonist L144 was able to induce a modest response to Q144 in Q144-tolerized 1B6 tg T cells, with preferential production of Th2 cytokines.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of immunization on the cytokine response of Q144-tolerized 1B6 T cells. Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with either PBS or 300 µg of Q144 in PBS i.p. 2 days later (Qtol). After 10 days, mice were immunized with either 100 µg of Q144 (Qtol/Qimm) or 10 µg of L144 in CFA (Qtol/Limm). As control, untolerized recipient mice were immunized with 100 µg of Q144 (Qimm) or 10 µg of L144 (Limm). Cells were isolated 5 days later and stimulated with 10 µg/ml ({blacksquare}) or 100 µg/ml ({square}) Q144. The cytokine response to Q144 was determined in supernatants of LNC cultures.

 
High-dose soluble superagonist L144 results in general unresponsiveness of 1B6 tg T cells

Because the superagonist ligand L144 induced responses in tolerized 1B6 tg T cells, and broke tolerance to cognate ligand, we asked whether a high-dose soluble superagonist would induce unresponsiveness to itself and to other 1B6 TCR ligands. Mice that received 1B6 tg T cells were subsequently tolerized with three different concentrations of L144 (3, 10, and 50 µg). Proliferation of isolated LNC was determined in response to 1 µg/ml L144 10 days later. Even the lowest dose of soluble L144 was able to induce a reduction of the proliferative response to L144, which was more pronounced with increasing doses of tolerogen (data not shown).

As described, L144 was able to break unresponsiveness induced by the weaker agonists Q144 or Y144, and we now asked whether L144-tolerized 1B6 tg T cells can respond to Q144 and Y144. Recipient SJL mice were tolerized with 10 µg of soluble L144, and LNC were restimulated with W144, Q144, Y144, and L144 peptides in vitro 10 days later. None of the tested peptides induced proliferation of 1B6 tg T cells (Fig. 5A). Similar results were obtained by tolerization with 100 µg of soluble L144 (data not shown). These data demonstrate that, once tolerance was induced by superagonist L144, LNC were unable to proliferate to any tested 1B6 TCR ligands, not even the superagonist L144. We next asked whether this unresponsiveness could be overcome by adding exogenous IL-2 to the T cell cultures, as demonstrated in the classical anergy experiments (17). However, addition of exogenous recombinant human IL-2 to the cultures was unable to break unresponsiveness induced by tolerization with L144 (Fig. 5B, left panel). In contrast, T cells proliferated when stimulated with increasing dosages of PMA (4–20 ng/ml) and ionomycin (0.08–0.4 µg/ml), suggesting that signaling events distal of the TCR were retained and not directly involved in L144-induced tolerance of 1B6 T cells (Fig. 5B, right panel).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. Response of 1B6 tg T cells following tolerization with soluble superagonist L144. A, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with 10 µg of soluble L144 i.p. 2 days later. LNC were isolated after 10 days from tolerized and nontolerized mice and control. Proliferation of LNC was determined in response to indicated concentrations of L144 (top axis) or W144, Q144, and Y1444 (bottom axis). B, Recipient mice were tolerized with 10 µg of L144, and LNC were isolated 10 days later. Proliferative responses to indicated concentrations of L144 in the presence of IL-2 (left panel) in medium or to PMA/ionomycin stimulation (right panel) was assessed. C, Purified 5 x 106 1B6 T cells were transferred into wild-type SJL/J mice. Recipient mice were injected with either L144 (1 or 10 µg) in CFA (immunized), PBS (untolerized), or soluble L144 (50 or 100 µg) (tolerized) 2 days later. Cytokine production was examined 10 days later in supernatants of LNC cultures stimulated with 100 µg/ml W144, Q144, or Y144 or 1 µg/ml L144 for 48 h.

 
Next, we examined whether LNC tolerized with superagonist were able to produce cytokines in response to different ligands. Mice were tolerized with 1 or 50 µg of soluble L144 or immunized with 1 or 10 µg of L144 in CFA. LNC were restimulated 10 days later with 100 µg/ml W144, Q144, and Y144 or with 1 µg/ml L144. Secretion of IL-2, IL-4, IL-10, and IFN-{gamma} was measured by ELISA in supernatants after 48 h. As shown in Fig. 5C, secretion of IL-2 and IFN-{gamma} was completely abrogated in response to all tested TCR ligands following tolerization with the L144 peptide. In contrast, immunization with L144 increased the IL-2 and IFN-{gamma} production of 1B6 tg T cells in response to Q144, Y144, and L144 in a dose-dependent and hierarchical manner. IL-4 and IL-10 cytokines were not detected. Taken together, although superagonist L144 was able to induce an altered hierarchical cytokine response in 1B6 tg T cells tolerized with cognate peptide, once these T cells were tolerized with L144, they were unable to produce Th1 or Th2 cytokines in response to any of the 1B6 TCR ligands tested.

Furthermore, tolerization with L144 resulted in an increased in vivo percentage (Fig. 1C) and cycling (data not shown) of tg 1.16+CD4+ T cells, indicating proliferation. These data suggested that 1B6 tg T cells were able to divide in response to tolerogen L144 in vivo, but that they were unresponsive when further activated on day 10 ex vivo. This might suggest that the global unresponsiveness induced by L144 requires active cell division by T cells (29).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
How tolerance to self-Ags is sustained, when responses to unrelated foreign Ags can activate autoreactive T cells, is one of the basic questions regarding autoimmunity. One possible mechanism is that tolerance to self results in unresponsiveness to foreign Ags (32). We have recently shown that a cross-reactive superagonist induced unresponsiveness of self-specific, untolerized T cells (29). In this study, we demonstrated how tolerance is maintained or broken by the same superagonist, L144, if the self-specific T cells had been already tolerized by the cognate peptide. We observed that, although superagonist could break tolerance to the cognate ligand, an anti-inflammatory cytokine production was induced.

Several studies have previously addressed whether tolerance to cognate peptides could be broken. Those data suggested that breaking tolerance involves stimulation of low-affinity clones, which escape tolerance induction (33, 34, 35). Recently, it has been suggested that anergy was not reversed on the clonal level, rather, breaking tolerance involved the activation of T cells with low affinity for the tolerogen and high affinity for the heteroclitic Ag (36, 37). In contrast, our data imply that superagonist L144 reversed tolerance at a clonal level. However, L144 is a superagonist peptide, which is capable of inducing maximal proliferation at 7 logs lower Ag concentration than tolerogenic ligand Q144. The agonist ligand Y144, which elicits responses at a 10-fold lower concentration than Q144, was unable to induce a response in Q144-tolerized tg T cells; this is consistent with previous reports (36). Our data suggest that a very strong stimulus provided by a superagonist is needed to reverse tolerance at the clonal level. Because the reversal of tolerance was also observed in tg 1B6 cells on the RAG2-deficient background, it excludes the possibility that untolerized low-avidity T cells expressing endogenous TCR {alpha}-chains, coupled with the tg {beta}-chain, responded to L144.

Furthermore, the reversal of clonal tolerance was characterized by the production of the Th2 cytokines IL-4 and IL-10 in response to subsequent activation with the cognate ligand. We observed the production of anti-inflammatory cytokines in two experimental systems. First, transgenic T cells tolerant to Q144 responded to the superagonist with the production of IL-4 in vitro. This response was similar to the previously observed Th2 cytokine production by T cells anergized with high-affinity ligand (38). In addition, in mice tolerized with Q144, immunization with L144 was able to abrogate tolerance and elicit a T cell response to Q144 in vitro. However, responding 1B6 T cells also produced IL-4 and IL-10 and significantly less IFN-{gamma} and IL-2 compared with untolerized mice. It has been shown previously that chronic Ag stimulus may result in anergy as well as in IL-10 production in vivo (39, 40, 41). The phenotype of 1B6 T cells after breaking tolerance was very similar to these previously described anergic T cells: they produced IL-10, but low levels of IL-2 and IFN-{gamma} in response to Q144 restimulation. This blunted and deviated immune response indicates that superantigen L144 does not restore the cognate response to Q144 but elicits an "altered" response in the tolerized cells, similar to that induced by repeated antigenic stimulation (39, 40, 41). It is likely that the stimulus provided by superagonist is weak in these tolerized cells and induces Th2 responses, which require low activating signals for activation similar to that induced by a suboptimal antigenic stimulation. Numerous studies have shown that a suboptimal stimulus provided by structural alterations (42, 43) or by changing the amount of an antigenic ligand (43, 44, 45, 46, 47) affects the polarized differentiation into Th1/Th2 effector cells such that suboptimal stimulus favors the production of Th2 cytokines. We may also speculate that IL-10 and IL-4 induced by L144 may be required for maintaining tolerance and suppressing inflammatory cross-reactive Th1 autoimmune responses (48, 49, 50). Taken together, our data suggest two main mechanisms that may prevent a detrimental autoreactive T cell response induced by a cross-reactive superagonist: 1) if autoreactive T cells are not tolerized, they may become unresponsive to the cognate self-ligand upon encounter with the superagonist, as we have described previously (29). 2) If T cells are already tolerized to self-Ag, a cross-reactive superagonist may induce a blunted response to the tolerizing ligand and production of anti-inflammatory Th2 cytokines. This altered response may counterbalance the "broken tolerance."

Because we observed that superagonist L144 could break unresponsiveness to cognate ligand Q144, we examined whether the superagonist itself is able to induce tolerance. In contrast to our expectations, we found that high-dose soluble L144 induced a general unresponsiveness to all tested 1B6 TCR ligands. Considering that L144 could elicit a response in T cells tolerized with Q144, but no response could be induced with any cross-reactive ligands when 1B6 cells were tolerized with L144, this implies that tolerance was induced in a hierarchical fashion. The in vivo expansion followed by in vitro unresponsiveness induced by L144 tolerization was very similar to tolerance induced by 50 µg of L144 immunization (29). It is possible that unresponsiveness induced by either high-dose soluble L144 (50–100 µg) or immunization with L144 in CFA (50 µg) may be mediated by similar mechanisms, because in both cases: 1) exogenous IL-2 failed to rescue the unresponsiveness; 2) the responses downstream of the TCR induced by PMA/ionomycin were retained; and 3) the L144-treated cells were accompanied with up-regulation of CD5 in vivo, which may be important in this kind of unresponsiveness as a negative signal (data not shown). It is also possible that soluble L144 might invoke different mechanisms for the induction of tolerance when compared with soluble Q144. In summary, if 1B6 tg T cells were not tolerized, the cross-reactive superagonist L144 induced Th1 cytokine production in naive T cells in vitro (29). If 1B6 cells were tolerized to the cognate ligand Q144, superagonist L144 elicited an altered but blunted response in vitro. Similarly, although immunization with low-dose superagonist was able to break tolerance to the cognate ligand Q144, this response was blunted and characterized by production of Th2 cytokines. This blunted response with altered cytokine production may provide a mechanism by which a cross-reactive high-affinity ligand is not able to induce a pathogenic autoimmune response in tolerized self-reactive T cell clones.


   Acknowledgments
 
We thank Robert McGilp and Deneen Kozoriz for the excellent FACS sorting at Center for Neurologic Diseases, Harvard Medical School, and Lea Cefalu and Nichole Price for animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from National Institutes of Health (RO1 NS30843, PO1 NS38037, and RO1 AI44880) and the National Multiple Sclerosis Society (2571-D9 to V.K.K. and RG 3257 to L.B.N.). V.K.K. is a recipient of Javitz Neuroscience Investigator Award. Back

2 Current address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139. Back

3 Current address: F44, School of Medical Sciences, University Walk, Bristol BS8 1TD, U.K. Back

4 Address correspondence and reprint requests to Dr. Vijay K. Kuchroo, Center for Neurologic Diseases, Harvard Institute of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. E-mail address: vkuchroo{at}rics.bwh.harvard.edu Back

5 Abbreviations used in this paper: tg, transgenic; PLP, proteolipid protein; LNC, lymph node cell. Back

Received for publication December 30, 2004. Accepted for publication May 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Anderson, A. C., L. B. Nicholson, K. L. Legge, V. Turchin, H. Zaghouani, V. K. Kuchroo. 2000. High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire. J. Exp. Med. 191: 761-770.[Abstract/Free Full Text]
  2. Klein, L., M. Klugmann, K. A. Nave, V. K. Tuohy, B. Kyewski. 2000. Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6: 56-61.[Medline]
  3. Liston, A., S. Lesage, J. Wilson, L. Peltonen, C. C. Goodnow. 2003. Aire regulates negative selection of organ-specific T cells. Nat. Immunol. 4: 350-354.[Medline]
  4. Bevan, M. J.. 1997. In thymic selection, peptide diversity gives and takes away. Immunity 7: 175-178.[Medline]
  5. Goldrath, A. W., M. J. Bevan. 1999. Selecting and maintaining a diverse T-cell repertoire. Nature 402: 255-262.[Medline]
  6. Kuchroo, V. K., A. C. Anderson, H. Waldner, M. Munder, E. Bettelli, L. B. Nicholson. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning and regulating the autopathogenic T cell repertoire. Annu. Rev. Immunol. 20: 101-123.[Medline]
  7. Ohashi, P. S., S. Oehen, K. Buerki, H. Pircher, C. T. Ohashi, B. Odermatt, B. Malissen, R. M. Zinkernagel, H. Hengartner. 1991. Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 65: 305-317.[Medline]
  8. Oldstone, M. B., M. Nerenberg, P. Southern, J. Price, H. Lewicki. 1991. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response. Cell 65: 319-331.[Medline]
  9. Schonrich, G., U. Kalinke, F. Momburg, M. Malissen, A. M. Schmitt-Verhulst, B. Malissen, G. J. Hammerling, B. Arnold. 1991. Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 65: 293-304.[Medline]
  10. Zhang, L., W. Fung-Leung, R. G. Miller. 1995. Down-regulation of CD8 on mature antigen-reactive T cells as a mechanism of peripheral tolerance. J. Immunol. 155: 3464-3471.[Abstract]
  11. Wang, R., Y. Wang-Zhu, H. Grey. 2002. Interactions between double positive thymocytes and high-affinity ligands presented by cortical epithelial cells generate double negative thymocytes with T cell regulatory activity. Proc. Natl. Acad. Sci. USA 99: 2181-2186.[Abstract/Free Full Text]
  12. Jones, L. A., L. T. Chin, D. L. Longo, A. M. Kruisbeek. 1990. Peripheral clonal elimination of functional T cells. Science 250: 1726-1729.[Abstract/Free Full Text]
  13. Carlow, D. A., S. J. Teh, N. S. van Oers, R. G. Miller, H. S. Teh. 1992. Peripheral tolerance through clonal deletion of mature CD4–CD8+ T cells. Int. Immunol. 4: 599-610.[Abstract/Free Full Text]
  14. Zhang, L. I., R. Martin, W. P. Fung-Leung, H. S. Teh, R. G. Miller. 1992. Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen. J. Immunol. 148: 3740-3745.[Abstract]
  15. Jenkins, M. K., R. H. Schwartz. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165: 302-319.[Abstract/Free Full Text]
  16. Mueller, D. L., M. K. Jenkins, R. H. Schwartz. 1989. Clonal expansion versus functional clonal inactivation: a costimulatory signaling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7: 445-480.[Medline]
  17. Schwartz, R. H.. 1990. A cell culture model for T lymphocyte clonal anergy. Science 248: 1349-1356.[Abstract/Free Full Text]
  18. Rocha, B., C. Tanchot, H. von Boehmer. 1993. Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen. J. Exp. Med. 177: 1517-1521.[Abstract/Free Full Text]
  19. Forster, I., R. Hirose, J. M. Arbeit, B. E. Clausen, D. Hanahan. 1995. Limited capacity for tolerization of CD4+ T cells specific for a pancreatic {beta} cell neoantigen. Immunity 2: 573-585.[Medline]
  20. Falb, D., T. J. Briner, G. H. Sunshine, C. R. Bourque, M. Luqman, M. L. Gefter, T. Kamradt. 1996. Peripheral tolerance in T cell receptor-transgenic mice: evidence for T cell anergy. [Published erratum appears in 1996 Eur. J. Immunol. 26: 966.]. Eur. J. Immunol. 26: 130-135.[Medline]
  21. Akkaraju, S., W. Y. Ho, D. Leong, K. Canaan, M. M. Davis, C. C. Goodnow. 1997. A range of CD4 T cell tolerance: partial activation to organ-specific antigen allows nondestructive thyroiditis or insulitis. Immunity 7: 255-271.[Medline]
  22. Rellahan, B. L., L. A. Jones, A. M. Kruisbeek, A. M. Fry, L. A. Matis. 1990. In vivo induction of anergy in peripheral V{beta}8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172: 1091-1100.[Abstract/Free Full Text]
  23. Blackman, M. A., T. H. Finkel, J. Kappler, J. Cambier, P. Marrack. 1991. Altered antigen receptor signaling in anergic T cells from self-tolerant T-cell receptor B-chain transgenic mice. Proc. Natl. Acad. Sci. USA 88: 6682-6686.[Abstract/Free Full Text]
  24. Migita, K., A. Ochi. 1993. The fate of anergic T cells in vivo. J. Immunol. 150: 763-770.[Abstract]
  25. Miethke, T., C. Wahl, H. Gaus, K. Heeg, H. Wagner. 1994. Exogeneous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship. Eur. J. Immunol. 24: 1893-1902.[Medline]
  26. Perkins, D. L., Y. Wang, S. Ho, G. R. Wiens, J. G. Seidman, I. J. Rimm. 1993. Superantigen-induced peripheral tolerance inhibits T cell responses to immunogenic peptides in TCR ({beta}-chain) transgenic mice. J. Immunol. 150: 4284-4291.[Abstract]
  27. Bar-Or, A., E. M. Oliveira, D. E. Anderson, D. A. Hafler. 1999. Molecular pathogenesis of multiple sclerosis. J. Neuroimmunol. 100: 252-259.[Medline]
  28. Olson, J.K., J. L. Croxford, M. A. Calenoff, M. C. Dal Canto, S. D. Miller. 2001. A virus-induced molecular mimicry model of multiple sclerosis. J. Clin. Invest. 108: 311-318.[Medline]
  29. Munder, M., E. Bettelli, L. Monney, J. M. Slavik, L. B. Nicholson, V. K. Kuchroo. 2002. Reduced self-reactivity of an autoreactive T cell after activation with cross-reactive non-self-ligand. J. Exp. Med. 196: 1151-1162.[Abstract/Free Full Text]
  30. Nicholson, L. B., H. Waldner, A. M. Carrizosa, A. Sette, M. Collins, V. K. Kuchroo. 1998. Heteroclitic proliferative responses and changes in cytokine profile induced by altered peptides: implications for autoimmunity. Proc. Natl. Acad. Sci. USA 95: 264-269.[Abstract/Free Full Text]
  31. Kearney, E., K. Pape, D. Loh, M. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1: 327-339.[Medline]
  32. Vidovic, D., P. Matzinger. 1988. Unresponsiveness to a foreign antigen can be caused by self-tolerance. Nature 336: 222-225.[Medline]
  33. Targoni, O. S., P. V. Lehmann. 1998. Endogeneous myelin basic protein inactivates the high avidity T cell repertoire. J. Exp. Med. 187: 2055-2063.[Abstract/Free Full Text]
  34. Poplonski, L., B. Vukusic, J. Pawling, S. Clapoff, J. Roder, N. Hozumi, J. Wither. 1996. Tolerance is overcome in beef insulin-transgenic mice by activation of low-affinity autoreactive T cells. Eur. J. Immunol. 26: 601-609.[Medline]
  35. Theobald, M., J. Biggs, J. Hernandez, J. Lustgarten, C. Labadie, L. A. Sherman. 1997. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J. Exp. Med. 185: 833-841.[Abstract/Free Full Text]
  36. Zügel, U., R. Wang, G. Shih, A. Sette, J. Alexander, H. M. Grey. 1998. Termination of peripheral tolerance to a T cell epitope by heteroclitic antigen analogues. J. Immunol. 161: 1705-1709.[Abstract/Free Full Text]
  37. Wang, R., Y. Wang-Zhu, C. R. Gabaglia, K. Kimachi, H. M. Grey. 1999. The stimulation of low-affinity, nontolerized clones by heteroclitic antigen analogues causes breaking of tolerance established to an immunodominant T cell epitope. J. Exp. Med. 190: 983-994.[Abstract/Free Full Text]
  38. Pearson, C. I., W. van Ewijk, H. O. McDewitt. 1997. Induction of apoptosis and T helper 2 (Th2) responses correlates with peptide affinity for the major histocompatibility complex in self-reactive T cell receptor transgenic mice. J. Exp. Med. 185: 583-599.[Abstract/Free Full Text]
  39. Buer, J., A. Lanoue, A. Franzke, C. Garcia, H. von Boehmer, A. Sarukhan. 1998. Interleukin 10 secretion and impaired effector function of major histocompatibility complex class II-restricted T cells anergized in vivo. J. Exp. Med. 187: 177-183.[Abstract/Free Full Text]
  40. Sundstedt, A., I. Höidén, A. Rosendahl, T. Kalland, N. van Rooijen, M. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J. Immunol. 158: 180-186.[Abstract]
  41. Bachetta, R., M. Bigler, J. L. Touraine, R. Parkman, P. A. Tovo, J. Abrams, M. G. Roncarolo. 1994. high levels of IL-10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoetic stem cells. J. Exp. Med. 179: 493-502.[Abstract/Free Full Text]
  42. Evavold, B. D., P. M. Allen. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science 252: 1308-1310.[Abstract/Free Full Text]
  43. Hemmer, B., I. Stefanova, M. Vergelli, R. N. Germain, R. Martin. 1998. Relationships among TCR ligand potency, thresholds for effector function elicitation, and the quality of early signaling events in human T cells. J. Immunol. 160: 5807-5814.[Abstract/Free Full Text]
  44. Hosken, N. A., K. Shibuya, A. W. Heath, K. M. Murphy, A. O’Gara. 1995. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-{alpha}{beta}-transgenic model. J. Exp. Med. 182: 1579-1584.[Abstract/Free Full Text]
  45. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, K. Bottomly. 1995. Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182: 1591-1596.[Abstract/Free Full Text]
  46. Tao, X., C. Grant, S. Constant, K. Bottomly. 1997. Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding. J. Immunol. 158: 4237-4244.[Abstract]
  47. Ise, W., M. Totsuka, Y. Sogawa, A. Ametani, S. Hachimura, T. Sato, Y. Kumagai, S. Habu, S. Kaminogawa. 2002. Naive CD4+ T cells exhibit distinct expression patterns of cytokines and cell surface molecules on their primary responses to varying doses of antigen. J. Immunol. 168: 3242-3250.[Abstract/Free Full Text]
  48. Mathisen, P. M., M. Yu, J. M. Johnson, J. A. Drazba, V. Tuohy. 1997. Treatment of experimental autoimmune encephalomyelitis with genetically modified memory T cells. J. Exp. Med. 186: 159-164.[Abstract/Free Full Text]
  49. Groux, H., A. O’Gara, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T cell subset inhibits antigen specific T cell responses and prevents colitis. Nature 389: 737-742.[Medline]
  50. Bettelli, E., M. P. Das, E. D. Howard, H. L. Weiner, R. A. Sobel, V. K. Kuchroo. 1998. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161: 3299-3306.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
Z. Illes, H. Waldner, J. Reddy, A. C. Anderson, R. A. Sobel, and V. K. Kuchroo
Modulation of CD4 co-receptor limits spontaneous autoimmunity when high-affinity transgenic TCR specific for self-antigen is expressed on a genetically resistant background
Int. Immunol., October 1, 2007; 19(10): 1235 - 1248.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. L. Kemp, L. Wille, C. L. Lewis, L. B. Nicholson, and D. A. Lauffenburger
Quantitative Network Signal Combinations Downstream of TCR Activation Can Predict IL-2 Production Response
J. Immunol., April 15, 2007; 178(8): 4984 - 4992.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Illés, Z.
Right arrow Articles by Kuchroo, V. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Illés, Z.
Right arrow Articles by Kuchroo, V. K.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS