The JI PBL Intereron Source
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 Related articles in The JI
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 Yu, P.
Right arrow Articles by Zaghouani, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, P.
Right arrow Articles by Zaghouani, H.
The Journal of Immunology, 2005, 174: 6772-6780.
Copyright © 2005 by The American Association of Immunologists

Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen1

Ping Yu*, Randal K. Gregg2,*, J. Jeremiah Bell*, Jason S. Ellis*, Rohit Divekar*, Hyun-Hee Lee*, Renu Jain*, Hanspeter Waldner3,{ddagger}, John C. Hardaway*, Mary Collins{dagger}, Vijay K. Kuchroo{ddagger} and Habib Zaghouani4,*

* Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212; {dagger} Wyeth Research, Cambridge, MA 02140; and {ddagger} Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F1 (SJL/J x C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the past 5 years, tremendous progress has been made toward understanding suppressive mechanisms of T regulatory (Treg)5 cells (1, 2, 3, 4, 5). However, most of the investigations relied on naturally occurring or disease-associated Treg cells for these functional characterizations. It is now well accepted that Treg cells maintain peripheral tolerance and regulate autoimmunity (6, 7, 8, 9, 10). However, circumstances under which this regulatory mechanism fails results in autoimmunity. Expansion of Treg cells is required to overcome the onset and progression of autoimmune disease. Recently, it has been shown that defined suppressive drugs (11, 12, 13) and hormones (14), as well as anti-CD3 Ab (15), can indeed expand Treg cells endowed with suppressive functions. However, these approaches may target both useful and pathogenic T cells limiting therapeutic application. Ag-specific strategies may circumvent these limitations, but approaches to expand specific Treg cells remains largely undefined.

Ig-proteolipid protein (PLP)1, an Ig molecule carrying the encephalitogenic PLP1 peptide corresponding to aa 139-151 of PLP efficiently internalizes into APCs via Fc{gamma}Rs and augments loading of PLP1 peptide onto MHC class II molecules (16). Consequently, presentation to T cells is enhanced by several orders of magnitude (16). Upon aggregation, Ig-PLP1 acquires the additional property, namely cross-linking of Fc{gamma}Rs, which result in IL-10 production by the APCs (17). Ultimately, aggregated (agg) Ig-PLP1 was able to reverse ongoing experimental allergic encephalomyelitis (EAE), whether induced by a single epitope or by CNS homogenate containing multiple encephalitogenic determinants (16, 17, 18). Given that amelioration of disease was dependent upon IL-10 and this cytokine serves as growth factor for Treg cells (19, 20), it is possible that agg Ig-PLP1 treatment mobilizes Treg cells for reversal of EAE. This study takes advantage of the agg Ig-PLP1/IL-10 system along with the PLP1-specific 5B6 TCR transgenic (Tg) mice (21, 22) to test whether agg Ig-PLP1 can induce expansion of Ag-specific Treg cells. Furthermore, the Treg cells were assessed for suppressive functions against EAE involving restricted as well as diverse T cell specificities. The findings indicate that in vivo exposure to adjuvant-free agg Ig-PLP1 drives naive 5B6 TCR Tg T cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3, which produce IL-10. Moreover, these 5B6 Treg cells display suppressive function and reversed passive EAE induced in RAG-2–/– SJL mice by transfer of 5B6 pathogenic T cells. Similarly, the 5B6 Treg cells reversed active EAE induced in the wild-type SJL/J mice with the cognate ligand PLP1 peptide. Interestingly, there was a broad efficacy against the disease, and these IL-10-producing Treg cells were able to display bystander suppression and reverse EAE induced in SJL/J mice with a CNS homogenate that likely incorporates multiple epitopes. However, intriguingly, the 5B6 Treg cells reversed EAE induced in F1 (SJL/J x C57BL/6) mice by the I-As-restricted cognate PLP1 peptide but could not display bystander suppression and protect the F1 animals against disease induced with the I-Ab-restricted myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (designated MOG peptide). The lack of bystander suppression was not due to strain restriction because the 5B6 Treg cells were unable to protect SJL/J mice against EAE mediated by myelin basic protein (MBP) 87-99 peptide (designated MBP3 peptide), which is restricted to the I-As class II allele. However, when the T cells were preactivated in vitro with PLP1-loaded APCs, they became able to reverse MOG-induced disease in the F1 similar to MBP3-induced EAE in SJL/J mice. Altogether, the results indicate that Treg cells would be able to overcome epitope spreading and reverse autoimmunity as long as the cognate Ag is available for activation.


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

SJL/J (H-2s) and C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory and bred and maintained in our animal care facility for the duration of the experiments. F1 mice were generated by crossing SJL/J males with C57BL/6 females. RAG-2-deficient (RAG-2–/–) SJL/J mice carrying PLP1-specific 5B6 TCR as transgene (H-2s background) were described previously (21). All experimental procedures were performed according to the guidelines of the institutional animal care committee.

Antigens

Peptides. The peptides used in this study were purchased from Metabion and purified by HPLC to >90% purity. PLP1 peptide (HSLGKWLGHPDKF) encompasses amino acid residues 139-151 of PLP (23), PLP2 peptide includes amino acid residues 178-191(NTWTTCQSIAFPSK) of PLP (24), and MBP3 peptide corresponds to amino acid residues 87-99 (VHFFKNIVTPRTP) of MBP (25). PLP1, PLP2, and MBP3 peptides are restricted to I-As and are encephalitogenic in SJL/J (H-2s) mice (23, 24, 25). MOG peptide (MEVGWYRSPFSRVVHLYRNGK) containing amino acid residues 35-55 of MOG is restricted to I-Ab and is encephalitogenic in C57BL/6 mice (26).

Ig chimeras. The Ig chimeras used in this study are described in Table I. All chimeras carry the corresponding peptide within the H chain CDR3 region. Ig-W is the parental Ig not encompassing any myelin or other peptide. All chimera transfectants were grown up in large-scale culture of DMEM containing 10% iron-enriched calf serum (HyClone). The chimeras were purified from culture supernatant on affinity chromatography columns made of rat anti-mouse {kappa}-chain coupled to CNBr-activated Sepharose 4B (Amersham Biosciences). To avoid cross-contamination, separate columns were used to purify each chimera. Aggregation of the Ig chimeras was done using 50%-saturated (NH4)2SO4 as described previously (17). Because all the Ig chimeras were derived from the same Ig backbone and thereby comprise identical IgG2b isotype, their Fc{gamma}Rs will be similar. In this respect, we may refer to them indistinguishably as Ig chimeras.


View this table:
[in this window]
[in a new window]
  		Table I. Ig chimeras used in this study

 
CNS homogenate. Frozen unstripped rat brains (Pelfreez Biologicals) were homogenized in PBS using a Waring blender and adjusted to 300 mg/ml with PBS as described previously (17).

Expansion and isolation of Treg cells

Expansion of specific Treg cells was conducted in two ways. SJL/J mice recipient of 5 x 106 splenic CD4+ T cells from RAG-2–/– 5B6 TCR Tg SJL mice were given an i.p. injection of 300 µg of agg Ig-PLP1 on days 4, 8, and 12 posttransfer. The mice were sacrificed, and their spleens were used for purification of Treg cells. For expansion of Treg cells in RAG-2–/– 5B6 TCR Tg SJL mice, three injections of 300 µg of agg Ig-PLP1 were given at 4-day intervals, and the splenic Treg cells were isolated 10 days after the final injection. In both regimens, the Treg cells were detectable at day 7, but higher levels of CD4+CD25+ were observed at day 10. Thus, purification of Treg cells from the spleen was performed on day 10 by depleting CD4– cells using Miltenyi Biotec’s CD4+ isolation kit, and the CD4+CD25+ T cells were isolated on anti-CD25 microbeads, according to Miltenyi Biotec’s instructions. The purity of cells is usually 90–95%.

Flow cytometry for analysis of the phenotype of Treg cells

Splenic CD4+ T cells were isolated by MACS using anti-CD4 microbeads, according to Miltenyi Biotec’s instructions, and then stained with PE-conjugated anti-CTLA-4 Ab (UC10-4F10-11) or control hamster IgG at 37°C for 2 h. Subsequently, the cells were incubated with FITC-conjugated anti-TCR-V{beta}6 (RR4-7), APC-conjugated anti-CD25 (PC61), and PE-Cy5-conjugated anti-CD4 (H129.19) Abs at 4°C for 30 min. The cells were then washed and analyzed using a FACSVantage flow cytometer and the CellQuest software (BD Biosciences). Dead cells were excluded based on their forward and side scatter profiles.

Real-time PCR analysis for Foxp3 expression

Total RNA was extracted from both CD4+CD25+ and CD4+CD25– T cells using TRIzol reagent. The relative mRNA levels of forkhead/winged helix transcription factor gene (Foxp3) was determined by real-time PCR using 300 ng of RNA, 0.5 µM Foxp3 or {beta}-actin primers, and the QuantiTect SYBR Green Real-Time PCR kit from Qiagen as described previously (30). The Foxp3 and {beta}-actin primers were described previously (31).

Measurement of IL-10 by ELISA

Cytokine secretion by Treg cells was performed by incubating the purified cells (5 x 104/well) on anti-CD3 Ab (2C11)-coated plates (10 µg/ml) and measuring IL-10 48 h later by ELISA, according to the standard protocol of BD Pharmingen. The capture Ab was JES5-2A5, and the biotinylated Ab was JES5-16E3. Recombinant mouse IL-10 was used in all experiments for construction of standard curves. The cytokine concentration in culture supernatants was interpolated from the linear portion of the standard curve. The use of anti-CD3 Ab instead of peptide and APCs to stimulate the purified Treg cells is to ensure that IL-10 is produced by the Treg cells rather than APCs.

Proliferation assay

Proliferation assays were performed as described previously (31). Briefly, proliferation of CD4+CD25– was assayed by incubating 200 x 103 cells with PLP1 peptide (30 µg/ml) and irradiated (3000 rad) SJL splenocytes as APCs for 3 days. One microcuries per well of [3H] thymidine was added during the last 15 h of incubation. The cells were then harvested on a Trilux 1450 Microbeta Wallac Harvester, and incorporated [3H]thymidine was counted using the Microbeta 270.004 software (Wallac).

Induction of EAE

Active EAE. This was done as described previously (17). Briefly, mice (6–8 wk old) were induced for EAE by s.c. injection in the footpads and at the base of the limbs of a 200-µl IFA/PBS (v/v) solution containing 6 mg of CNS homogenate and 200 µg of Mycobacterium tuberculosis H37Ra (Difco). Six hours later, the mice were given i.v. 200 ng of purified Bordetella Pertussis toxin (List Biological Laboratories). A second injection of B. Pertussis toxin was given after 48 h. The mice were then scored daily for clinical signs of EAE as follows: 0, no clinical score; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb paralysis; and 5, moribund or death.

A similar protocol was used for induction of active EAE with PLP1, PLP2, MBP3, and MOG peptides, which were used at 100, 100, 200, and 300 µg/injection, respectively. Pertussis toxin was used at 75 ng for PLP1 or PLP2 peptide, 200 ng for MBP3 peptide, and 500 ng for MOG peptide.

Passive EAE. Six- to 8-week-old RAG-2–/– SJL/J mice were induced for EAE by transfer of 1 x 106 naive 5B6 TCR Tg T cells. These cells do not require activation before transfer into the RAG-2–/– SJL/J hosts. Also, Pertussis toxin is not required for the development of EAE.

Suppression of EAE with Treg cells

For active EAE 1 x 106 purified CD4+CD25+ T cells were adoptively transferred into SJL/J mice 3 days after or 1 day before induction of EAE as indicated. For suppression of passive EAE, 0.5 x 106 CD4+CD25+ T cells were given to RAG-2–/– SJL mice i.v. along with naive 5B6 TCR Tg T cells. Thereafter, the animals were monitored for clinical signs of EAE.

For neutralization of IL-10 during disease suppression by Treg cells, the SJL/J mice received CD4+CD25+5B6 Treg cells 1 day before induction of EAE with PLP1 peptide and were given i.p. 300 µg/mouse of purified anti-IL-10 Ab (JESS-2A5) on days 9, 13, and 17 postdisease induction.

Treatment of EAE with agg Ig chimeras

For treatment of disease with agg Ig chimeras, the mice were induced for EAE with CNS homogenate and then given i.p. 300 µg/mouse agg Ig chimera in 300 µl of PBS on days 9, 13, and 17 postdisease induction. The mice then continued to be monitored for clinical signs of EAE.

Depletion of CD25+ T cells in vivo

For depletion of CD25+ T cells, the mice were given i.p. two injections of anti-CD25 Ab (PC61) 4 and 2 days before the induction of disease with 6 mg of CNS homogenate. Each injection consisted of 500 µl of PBS containing 1 mg of anti-CD25 Ab. A group of mice received 1 mg of rat IgG instead of anti-CD25 Ab to serve as control. The animals were then subjected to agg Ig chimera treatment regimen as described above.

Activation of Treg cells

For activation of Treg cells before injection to animals, the cells (0.5 x 106 cells/ml) were incubated with irradiated (3000 rad) SJL splenocytes (1.5 x 106 cells/ml) and 30 µg/ml PLP1 peptide. After 24 h, the excess peptide was washed out, and the activated Treg cells were injected into mice.

Statistical analysis

All analysis for statistically significant differences was performed with Student’s paired t test. A value of p < 0.05 is considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Exposure to agg Ig-PLP1 drives PLP1-specific 5B6 TCR Tg T cells into Treg cells

SJL/J mice recipient of PLP1-specific 5B6 TCR (V{beta}6+) Tg T cells and treated with agg Ig-PLP1 increase both CD25 and CTLA-4 expression on their V{beta}6+ T cells (Fig. 1). In fact, the number of V{beta}6+CD4+ cells expressing CD25 was 6–8% in untreated or sol Ig-PLP1-treated 5B6-recipient SJL mice but increased to 13.6% in animals given agg Ig-PLP1. Similarly, CTLA-4 expression on V{beta}6+CD4+CD25+ cells rose from 6 to 10% in untreated or sol Ig-PLP1-treated 5B6-recipient SJL mice to 41% in the mice treated with the agg Ig-PLP1. Of note, the number of V{beta}6+CD4+ T cells was similar in SJL and SJL/5B6 mice despite the fact that the latter received 5 x 106 5B6 cells. This may be due to T cell homeostasis.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 1. PLP1-specific 5B6 TCR Tg T cells expand into CD25+CTLA-4+ lymphocytes upon exposure to agg Ig-PLP1 in vivo. PLP1-specific 5B6 TCR Tg CD4+ T cells were injected (5 x 106 cells/mouse) i.v. through the tail vein into adult SJL mice, and the recipients were given an i.p. injection of 300 µg of sol (SJL/5B6/Sol Ig-PLP1) or agg (SJL/5B6/Agg Ig-PLP1) Ig-PLP1 on days 4, 8, and 12 after the transfer. Ten days later, the splenic CD4+ T cells were isolated by positive selection on anti-CD4 Ab coupled to microbeads (Miltenyi Biotec), and the cells (2 x 106 cells/sample) were analyzed for cell surface expression of TCRV{beta}6 (left panel) or coexpression of TCRV{beta}6 and CD25 (median panel) or CD25 and CTLA-4 (right panel) by flow cytometry. A group of unmanipulated SJL mice (SJL) and a group of animals recipient of T cells transfer, but no Ig-PLP1 treatment (SJL/5B6) was included for control purposes. Cells were gated based on light scatter properties, and live cells were analyzed for TCR V{beta}6 expression (left panel). CD25 expression (median panel) was then assessed on the V{beta}6-positive cells. CTLA-4 expression (right panel) was examined on V{beta}6/CD25 double-positive cells. The numbers indicate the percentage of cells expressing the specific marker among total cells. The results are representative of five different experiments.

 
Similar results were obtained when agg Ig-PLP1 treatment was applied to the RAG-2–/– 5B6 TCR Tg SJL/J mice (Table II). Indeed, in the untreated mice, 8.8% of V{beta}6+CD4+ T cells expressed CD25, and 6.7% of these V{beta}6+CD4+CD25+ had CTLA-4. In contrast, the mice treated with agg Ig-PLP1 increased their CD25 expression to 21.3%, and among these V{beta}6+CD4+CD25+ cells, 42.2% had CTLA-4 on the surface. Overall, these results indicate that treatment with agg Ig-PLP1 increases CD25 and CTLA-4 expression on PLP1-specific TCR Tg T cells in vivo.


View this table:
[in this window]
[in a new window]
  		Table II. Treatment of RAG-2–/– 5B6 TCR Tg mice with agg Ig-PLP1 drives PLP1-specific CD4 T cells to evolve as regulatory cells expressing both CD25 and CTLA-4

 
The Ag-specific V{beta}6+CD4+CD25+ were purified and tested for Foxp3 mRNA expression. Fig. 2A shows that cells isolated from 5B6 Tg or SJL mice recipient of 5B6 T cells (SJL/5B6) had approximately a 4-fold increase in Foxp3 mRNA expression, whereas V{beta}6+CD4+CD25– counterparts had no such increase. Also, the cells produced significant amounts of IL-10 relative to the CD25– counterparts when they were stimulated with plate-bound anti-CD3 Ab (Fig. 2B). Stimulation with rat IgG isotype control did not lead to increased IL-10 production. Finally, when the V{beta}6+CD4+CD25+ cells were stimulated with PLP1 peptide presented on SJL splenic APCs, proliferation was minimal relative to their V{beta}6+CD4+CD25– counterparts (Fig. 2C). Interestingly, when both types of cells were mixed together and stimulated with PLP1 peptide, the proliferation of the V{beta}6+CD4+CD25– cells was reduced to minimal levels, indicating suppression of their responses by the V{beta}6+CD4+CD25+ cells. Taken together, these results indicated that agg Ig-PLP1 drives Ag-specific TCR Tg 5B6 T cells to acquire a phenotypic and functional characteristic signature of Treg cells.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. Agg Ig-PLP1-expanded CD4+CD25+ T cells produce IL-10 and suppress proliferative responses of their CD4+CD25– counterparts. Purified PLP1-specific CD4+CD25+ T cells tested for expression of Foxp3 mRNA expression (A), IL-10 production (B), and suppression of their CD4+CD25– counterparts (C). CD4+CD25+ and CD4+CD25– cells were purified from the spleens of agg Ig-PLP1-treated 5B6 Tg mice (5B6 Tg) or SJL animals recipient of 5B6 CD4 T cells (SJL/5B6). Each group included five mice. RNA was extracted from 1 x 105 cells by the TRIzol method, and 300 ng were used for analysis of Foxp3 expression by comparative real-time PCR. Because Foxp3 expression was similar in both populations, only cells from 5B6 Tg mice were used for IL-10 and proliferation assays. IL-10 production assays used 2 x 105 cells that were stimulated with plate-bound anti-CD3 Ab or rat IgG isotype control (10 µg/ml) for 48 h. Measurement of IL-10 was conducted by ELISA. The proliferation assays in C used 2 x 105 T cells and 1 x 105 SJL-irradiated (3000 rad) splenocytes as APCs. For coculture assays, the T cell number was 1 x 105 for CD4+CD25+ T cells and 2 x 105 CD4+CD25– cells. Stimulation used 20 µM PLP1 peptide, and the proliferation was assessed by [3H]thymidine incorporation after 72 h of incubation. Each bar represents the mean ± SD of triplicates. The results are representative of three experiments. These phenotypic and functional properties are characteristic features of Treg cells. Therefore, we will refer to these cells as 5B6 Treg cells.

 
Ag-specific Treg cells suppress both active and passive EAE

Because the agg Ig-PLP1-expanded 5B6 TCR Tg Treg cells were able to suppress their CD25– counterparts in vitro, we sought to test whether such suppressive functions could be operative against EAE induced by PLP1 peptide and thus mediated by polyclonal PLP1-specific pathogenic T cells. As can be seen in Fig. 3, transfer of 5B6 TCR Tg Treg cells 3 days after induction of EAE had minimal effects on the severity of the initial phase of disease relative to mice that did not receive any transfer (Fig. 3A). However, the clinical relapses usually associated with PLP1-induced EAE, which were evident in the mice recipient of no transfer, were not observed for the 60-day monitoring period. Moreover, when the transfer was performed 1 day before disease induction, the initial phase of EAE was reduced significantly with a mean maximal score of 1.1 ± 0.7, and the relapses were completely inhibited (Fig. 3B). This is a significant protection against the disease because mice without Treg transfer had a mean maximal clinical score of 3.0 ± 0.5 during the acute phase of EAE and had clinical relapses typical of PLP1-induced EAE.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. 5B6 Treg cells ameliorate both passive and active EAE in SJL/J mice. 5B6 Treg cells were purified from agg Ig-PLP1-treated 5B6 Tg mice, and 1 x 106 cells were adoptively transferred into SJL/J mice 3 days after (A) or 1 day before (B) induction of EAE with PLP1 peptide. A group of mice that did not receive 5B6 Treg transfer (circles) was included as control. For suppression of passive EAE (C), RAG-2–/– SJL mice were given i.v. 1 x 106 naive 5B6 TCR Tg T cells alone (circles) or together with 0.5 x 106 5B6 Treg cells (triangle), and the animals were monitored for clinical signs of EAE. D, One group of SJL mice (CD25+) received 1 x 106 5B6 Treg cells 1 day before EAE induction with PLP1 peptide. Another group (CD25+ + anti-IL-10) received 1 x 106 5B6 Treg cells 1 day before EAE induction with PLP1 peptide followed by an i.p. injection of anti-IL-10 Ab (300 µg/mouse) on day 9, 13, and 17 postdisease induction. A group of mice (Nil) that did not receive Treg cells or anti-IL-10 was included as control. Each point represents the mean clinical score of five to six mice.

 
It has been shown previously that naive 5B6 TCR Tg SJL/J mice develop spontaneous EAE (21). In the present study, we found that transfer of naive 5B6 TCR Tg T cells into RAG-2–/– SJL mice induces severe EAE that ultimately leads to animal death (Fig. 3C). This passive EAE system was then used to test the 5B6 TCR Tg Treg cells for suppressive functions. As can be seen in Fig. 3C, the mice recipient of naive 5B6 TCR Tg T cells had a disease onset at day 12 posttransfer with a rapid heightening of the clinical scores, leading to 33% mortality and a mean maximal score of 4.0 ± 0.5. The animals were unable to recover by day 70 and were thus sacrificed. Transfer of CD4+CD25+ 5B6 TCR Tg Treg cells into these mice significantly reduced the severity of the disease, and the mean maximal score was 1.6 ± 1.1. Also, no death was observed in this group of mice, and recovery was achieved by day 20 after T cell transfer. The suppressive functions of 5B6 Treg cells seem to be dependent partially on IL-10 as mice recipient of Treg cells 1 day before induction of EAE and given anti-IL-10 Ab restored the severity of their acute disease phase relative to animals that did not receive anti-IL-10 Ab (Fig. 3D) or mice that received Treg cells and rat IgG isotype control (data not shown). Overall, these results indicate that agg Ig-PLP1-expanded 5B6 Treg cells display effective suppressive functions against both active and passive EAE.

Treg cells induced by a single epitope reverse EAE involving diverse specificities

Experiments were set up to determine whether expansion of protective Treg cells is unique to Ig-PLP1 or other chimeras carrying different myelin epitopes could also expand similar protective Treg cells. PLP2 and MBP3 peptides were chosen for these studies because of their encephalitogenicity in SJL/J mice and the fact that the respective Ig-PLP2 and Ig-MBP3 chimeras are available. However, given that TCR Tg mice are not available for PLP2 and MBP3 peptides, we opted to test Ig-PLP2 and Ig-MBP3 rather directly and assessed whether suppression of disease by the chimeras is dependent on Treg cells. Furthermore, because the Treg cells expanded by Ig-PLP1 produce IL-10, bystander suppression could be operative, and protection should be possible against disease involving diverse T cell specificities. Thus, the chimeras were tested initially for amelioration of EAE induced with a CNS homogenate incorporating multiple T cells epitopes and presumably involving diverse T cells specificities. As can be seen in Fig. 4, mice induced for EAE with a CNS homogenate had a severe phase of paralysis with a mean maximal score of 2.4 ± 0.5, and the control agg Ig-W, the parental chimera not encompassing any myelin peptide, did not have any effect on ameliorating the disease. Treatment with agg Ig-PLP1 reduced the mean maximal score to 1.5 ± 0.4, and full recovery was achieved by day 25 postdisease induction (Fig. 4B). This effect is specific because Ig-W had no significant effects on the disease, and the clinical signs of EAE were similar to those observed in untreated (Nil) animals (Fig. 4A). Ig-PLP2 (containing PLP178-191) as well as Ig-MBP3 (incorporating MBP87-99) had significant effects on the disease and reduced the severity of EAE in a fashion similar to agg Ig-PLP1 (Fig. 4, C and D). Indeed, the mean maximal disease score was reduced from 2.4 ± 0.5 in the untreated group to 1.4 ± 0.4 and 1.4 ± 0.5 in mice treated with agg Ig-PLP2 and agg Ig-MBP3, respectively. Also, in both groups, recovery from the initial phase of disease was achieved on day 20 postdisease induction for agg Ig-PLP2-treated mice and on day 22 for those given Ig-MBP3.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 4. Aggregated Ig chimeras carrying a single antigenic determinant modulate disease involving multiple epitopes. Groups of SJL/J mice (six per group) were induced for EAE with 6 mg of CNS homogenate and on days 9, 13, and 17 and treated i.p. with 300 µg/injection of agg Ig-W (A), agg Ig-PLP1 (B), agg Ig-PLP2 (C), or agg Ig-MBP3 (D). A group of untreated mice (Nil) was included for comparison purposes. Each point represents the mean clinical score of six mice.

 
To assess for involvement of Treg cells in the protection against EAE, the SJL/J mice were given anti-CD25 Ab to deplete Treg cells before EAE induction with CNS homogenate and then treated with agg Ig chimera and monitored for clinical signs of EAE. The results indicate that depletion of Treg cells by anti-CD25 Ab nullifies the suppressive function of Ig-PLP1 (Fig. 5A), Ig-PLP2 (Fig. 5B), and Ig-MBP3 (Fig. 5C), and the disease was restored to full severity like untreated mice. Rat IgG used instead of anti-CD25 Ab did not restore disease severity, and the clinical signs of EAE were reduced by treatment with the Ig-PLP1 (Fig. 5A). In fact, the mean maximal clinical score in this group was ~1.4 like the undepleted but Ig-PLP1 treated mice (compare with Fig. 4). Overall, these results indicate that Treg cells are required for modulation of disease involving diverse T cell specificities and raise the question of how Treg cells expanded by a single epitope display broad suppression against diverse disease.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. Depletion of CD25+ T cells before disease induction with CNS homogenate abrogates amelioration of EAE by agg Ig chimeras. SJL/J mice (five per group) were given i.p. 1 mg of anti-CD25 Ab (triangles) twice (4 and 2 days) before EAE induction with CNS homogenate. The animals were then treated i.p. with 300 µg of agg Ig-PLP1 (A), Ig-PLP2 (B), or Ig-MBP3 (C) on days 9, 13, and 17 postdisease induction. A group of mice recipient of rat IgG instead of anti-CD25 (circles) and treated with agg Ig-PLP1 after EAE induction with CNS homogenate was included for control purpose. Also, a group of mice that was not depleted with anti-CD25 Ab and not treated with any Ig chimeras after induction of EAE (Nil) was included to serve as control. Each point represents the mean clinical score of five mice.

 
Requirement for Ag-induced activation for Treg cells to display intermolecular bystander suppression

To investigate the mechanism underlying broad suppression of disease by Treg cells, we began by testing I-As-restricted, PLP1-specific 5B6 Treg cells for suppression of intermolecular EAE induced by I-Ab-restricted MOG peptide. Accordingly, 5B6 Treg cells were expanded in RAG-2–/– 5B6 TCR Tg SJL/J mice with agg Ig-PLP1, transferred into F1 (SJL/J x C57BL/6) mice, and tested for suppression of EAE induced 1 day later in the hosts by I-Ab-restricted MOG peptide. Fig. 6 shows that transfer of 5B6 TCR Tg Treg cells significantly reduced the severity of PLP1-induced EAE in the F1 mice (Fig. 6A). Indeed, while the recipients of Treg cells had a mean maximal clinical score of 1.0 ± 0.4 and fully recovered by day 30 postdisease induction, those who did not receive transfer of Treg cells had a 3.1 ± 0.2 mean maximal score and did not fully recover for the 60-day clinical monitoring period. In contrast, when the F1 mice were induced for EAE with MOG peptide, the 5B6 Treg cells had no effect on the disease, and the mice had similar pattern of clinical EAE as those without Treg transfer (Fig. 6B). These results indicate that suppressive functions by Treg cells operate in an Ag-restricted manner. This observation was surprising given that Treg cells produce IL-10, which should be able to mediate bystander suppression. Given the possibility that genetic dilution of I-As may interfere with Ag presentation and Treg function, we sought to test the 5B6 Treg cells for suppression of EAE induced by an intramolecular PLP2 peptide as well as an intermolecular MBP3 peptide but in the SJL/J rather than F1 mice. Accordingly, SJL/J mice were given PLP1-specific 5B6 TCR Tg Treg cells and 1 day later induced for EAE with PLP2 or MBP3 peptide or CNS homogenate and monitored for clinical signs of EAE. As can be seen in Fig. 7, transfer of PLP1-specific Treg cells reduced the severity of EAE when the disease was induced by CNS homogenate or PLP2 peptide (Fig. 7, A and B). However, when MBP3 peptide was used for EAE induction, there was only minimal reduction in disease severity (Fig. 7C). Thus, Treg cells display intra- (PLP2) but not intermolecular (MBP3 and MOG) suppressive functions unless the specific Ag was provided during disease induction, as is the case for CNS homogenate.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. Expanded 5B6 TCR Tg Treg cells suppress PLP1- but not MOG-induced EAE in F1 (SJL/J x C57BL/6) mice. Seven-wk-old F1 (SJL/J x C57BL/6) mice were induced for EAE with either PLP1 (A) or MOG peptide (B) 1 day after transfer of 0.5 x 106 agg Ig-PLP1-expanded 5B6 TCR Tg Treg cells (CD25+). The mice were then scored daily for clinical signs of EAE. Animals that did not receive any T cell transfer (Nil) were included for control purpose. Each point represents the mean clinical score of five to six mice.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 7. 5B6 TCR Tg Treg cells suppress CNS homogenate- but not MBP3-induced EAE. SJL/J mice were induced for EAE with either CNS homogenate (A), PLP2 (B), or MBP3 (C) peptide 1 day after transfer of 0.5 x 106 agg Ig-PLP1-expanded 5B6 TCR Tg Treg cells (CD25+). The mice were then scored daily for clinical signs of EAE. Animals that did not receive any T cell transfer (Nil) were included for control purpose. Each point represents the mean clinical score of five to six mice.

 
These observations suggest that activation with a specific Ag may be required for Treg cells to exercise broad suppressive functions. To test this postulate, 5B6 TCR Tg Treg cells were activated in vitro by stimulation with PLP1 peptide presented on splenic APCs, transferred to mice, and the hosts were induced for EAE and monitored for clinical signs of the disease. As can be seen in Fig. 8, the activated Treg cells have become effective not only against MBP3-induced EAE in SJL/J mice but also against MOG-induced disease in F1 animals. Indeed, the mean maximal score of disease severity in SJL/J mice induced for EAE with MBP3 peptide was reduced from 2.3 ± 0.5 to 0.9 ± 0.4 when the animals received preactivated Treg cells (Fig. 8B and Table III). This is much more significant than the reduction observed with nonactivated Treg cells (Table III). Also, the mean maximal score of disease severity was decreased from 2.8 ± 0.5 to 1.8 ± 0.5 when the F1 mice induced for EAE with MOG peptide received the preactivated 5B6 Treg cells (Fig. 8D and Table III). This is also more significant than the reduction of severity obtained with nonactivated Treg cells (Table III). In fact, the animals recovered on day 30 postdisease induction in the case of preactivated Treg transfer while recovery was not achieved when the mice received no Treg transfer or transfer of nonactivated Treg cells (Table III). The preactivation of Treg cells had less effect on CNS as well as PLP2-induced EAE relative to nonpreactivated Treg cells. As such, in the animals induced with the CNS homogenate, the mean maximal score of disease severity was 1.8 ± 0.4 with nonpreactivated Treg cells and decreased to 1.6 ± 0.5 when the Treg cells were preactivated (Table III). Similarly, when EAE was induced with PLP2 peptide, the mean maximal score was 1.8 with nonpreactivated Treg cells and decreased to 1.2 ± 0.8 when the Treg cells were preactivated in vitro. Taken together, these results indicate that activation of the Treg cells is required for broad suppressive function.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 8. 5B6 TCR Tg Treg cells require Ag-induced activation to display bystander suppression. Purified agg Ig-PLP1-expanded 5B6 TCR Tg Treg cells were stimulated with 30 µg/ml PLP1 peptide presented on irradiated SJL/J APC splenocytes, and 24 h later, 1 x 106 activated Treg cells were transferred into SJL/J (A–C) or (SJL/J x C57BL/6) F1 (D) mice. Three days later, the mice were induced for EAE with 6 mg of CNS homogenate (A), 200 µg of MBP3 (B), 100 µg of PLP2 (C), and 300 µg of MOG peptide (D). The animals were then scored daily for clinical signs of EAE. Animals that did not receive any T cell transfer (circles) were included for control purpose. Each point represents the mean clinical score of five to six mice.

 

View this table:
[in this window]
[in a new window]
  		Table III. Activated Treg cells suppress EAE involving intermolecular epitopesa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The studies presented in this article defined an approach for expansion of Ag-specific Treg cells and explored the requirements for broad suppressive functions against autoimmunity involving diverse pathogenic T cell specificities. The findings indicated that injection of adjuvant-free agg Ig-PLP1 into mice drives 5B6 TCR Tg T cells into Treg cells expressing CD4, CD25, and CTLA-4 (Fig. 1 and Table II). Moreover, these cells had increased FoxP3 mRNA were nonproliferative and produced IL-10 (Fig. 2). This Treg phenotype is supported by a display of suppressive functions as proliferation of CD4+CD25– TCR Tg 5B6 counterparts was reduced significantly by the CD4+CD25+ 5B6 cells (Fig. 2). Taken together, these results indicate that exposure to adjuvant-free agg Ig-PLP1 drives 5B6 TCR Tg T cells to differentiate into IL-10-producing Treg cells. The soluble form of Ig-PLP1 was less effective in expanding 5B6 Treg cells, and this likely suggests that IL-10 produced by APCs upon Fc{gamma}R cross-linking by agg Ig-PLP1 (17) contributed to the expansion of Treg cells. This is in good agreement with prior studies suggesting that IL-10 serves as growth factor for development of Treg cells (19, 20). Furthermore, these PLP1-specific 5B6 TCR Tg Treg cells suppress both active and passive EAE involving a single (PLP1) T cell specificity, and such function was IL-10 dependent (Fig. 3). However, surprisingly, the 5B6 TCR Tg Treg cells were unable to ameliorate MOG-induced EAE in F1 mice or MBP3-mediated EAE in SJL/J animals despite the ability of suppressing PLP2- or even CNS homogenate-induced EAE (Figs. 6 and 7). These observations have led us to believe that Ag-induced activation is required for the 5B6 Treg cells to display broad suppressive functions. Indeed, induction of disease with CNS homogenate would activate diverse T cells, leading to exposure of most myelin proteins, including PLP. In this case, PLP1 peptide would be available and the Treg cells would be activated, produce IL-10, and in turn display bystander suppression. Similarly, induction of disease with PLP2 peptide would also expose PLP, which encompasses PLP1 peptide and thereby triggers activation of the Treg cells. However, exposure and presentation of PLP1 peptide may not occur when the disease is induced by MOG or MPB3 peptide, particularly because epitope spreading (32) has been shown to follow a sequential order (33). When these PLP1-specific Treg cells were activated in vitro by stimulation with PLP1 presented on splenic APCs, suppression of MOG-EAE in F1 mice as well as MBP3-EAE in SJL/J mice was observed (Fig. 8 and Table III). These results agree with previous report, suggesting that activation would be required for in vivo Treg-suppressive function (34). Also, the data presented in Fig. 4 showing that the severity of CNS-induced EAE can be reduced by diverse chimeras carrying a single myelin epitope support the notion that activation is required for Treg cells to display broad suppressive function. The likely scenario here could be that during induction of disease with CNS homogenate pathogenic and regulatory T cells are triggered. Treatment with the chimeras under noninflammatory circumstances would sustain activation and expansion of Treg cells but not pathogenic cells. Consequently, cytokine production by Treg cells is in motion promoting bystander suppression of disease involving multiple pathogenic specificities. In fact, when anti-CD25 Ab was injected to deplete Treg cells before induction of disease, the treatment with the Ig chimeras failed to reduce the severity of disease (Fig. 5).

The first point to be taken from these studies is that a regimen was defined that is as effective as other strategies (35, 36) for expansion of Treg cells but would be rather practical for amelioration of autoimmunity. Moreover, because the regimen involves Ags, the Treg cells would be of defined specificity rather than polyclonally activated cells (11, 12, 13, 15). The second point is that reactivation of Treg cells triggers cytokine production and overrides restriction for specificity. This may be the underlying mechanism for the controversial observations regarding specificity of Treg-suppressive function (14, 34, 37, 38). Also, a similar mechanism could be responsible for the differential Treg function associated with their activation status (39). Finally, the studies parallel with other observations demonstrating the potency of Treg cells expanded with a single epitope against autoimmunity involving diverse T cell specificities (40, 41, 42).

Agg Ig-peptide chimeras provide a means to expand Treg cells with specificity, which hitherto has not been possible, and convert potentially pathogenic T cells into a regulatory T cells as exemplified by the effect of agg Ig-PLP1 on the encephalitogenic TCR Tg 5B6 T cells.


   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 National Institutes of Health Grants 2RO1 NS37406 and RO1 AI48541 (to H.Z.). Back

2 Current address: Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908-1386. Back

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

4 Address correspondence and reprint requests to Dr. Habib Zaghouani, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, M616 Medical Sciences Building, Columbia, MO 65212. E-mail address: zaghouanih{at}health.missouri.edu Back

5 Abbreviations used in this paper: Treg, T regulatory; PLP, proteolipid protein; EAE, experimental allergic encephalomyelitis; Tg, transgenic; MOG, myelin oligodendrocyte glycoprotein; MBP, myelin basic protein; agg, aggregated. Back

Received for publication January 1, 2005. Accepted for publication March 21, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shevach, E. M.. 2000. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400.
  2. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune response. Annu. Rev. Immunol. 22: 531-562.[Medline]
  3. von Herrath, M. G., L. L. Harrison. 2003. Antigen-induced regulatory T cells in autoimmunity. Nat. Rev. Immunol. 3: 223-232.[Medline]
  4. Bluestone, J. A., A. K. Abbas. 2003. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3: 253-257.[Medline]
  5. O’Garra, A., P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat. Med. 10: 801-805.[Medline]
  6. Mottet, C., H. H. Uhlig, F. Powrie. 2003. Cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170: 3939-3943.[Abstract/Free Full Text]
  7. Madakamutil, L. T., I. Maricic, E. Sercarz, V. Kumar. 2003. Regulatory T cells control autoimmunity in vivo by inducing apoptotic depletion of activated pathogenic lymphocytes. J. Immunol. 170: 2985-2992.[Abstract/Free Full Text]
  8. Ehrenstein, M. R., J. G. Evans, A. Singh, S. Moore, G. Warnes, D. A. Isenberg, C. Mauri. 2004. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF-{alpha} therapy. J. Immunol. 200: 277-285.
  9. Tang, Q., K. J. Henriksen, M. Bi, E. B. Finger, G. Szot, J. Ye, E. L. Masteller, H. McDevitt, M. Bonyhadi, J. A. Bluestone. 2004. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199: 1455-1465.[Abstract/Free Full Text]
  10. Reddy, J., Z. Illes, X. Zhang, J. Encinas, J. Pyrdol, L. Nicholson, R. A. Sobel, K. W. Wucherpfennig, V. K. Kuchroo. 2004. Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. USA 101: 15434-15439.[Abstract/Free Full Text]
  11. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, A. O’Garra. 2002. In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195: 603-616.[Abstract/Free Full Text]
  12. Gregori, S., N. Giarratana, S. Smiroldo, M. Uskokovic, L. Adorini. 2002. A 1{alpha},25-dihydroxyvitamin D3 analog enhances regulatory cells and arrests autoimmune diabetes in NOD mice. Diabetes 51: 1367-1374.[Abstract/Free Full Text]
  13. Vieira, P. L., J. R. Christensen, S. Minaee, E. J. O’Neill, F. J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D. C. Wraith, A. O’Garra. 2004. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol. 172: 5986-5993.[Abstract/Free Full Text]
  14. Polanczyk, M. J., B. D. Carson, S. Subramanian, M. Afentoulis, A. A. Vandenbark, S. F. Ziegler, H. Offner. 2004. Estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment. J. Immunol. 173: 2227-2230.[Abstract/Free Full Text]
  15. Belghith, M., J. A. Bluestone, S. Barriot, J. Megret, J. Bach, L. Chatenoud. 2003. TGF-{beta}-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat. Med. 9: 1202-1208.[Medline]
  16. Legge, K. L., B. Min, N. T. Potter, H. Zaghouani. 1997. Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing. J. Exp. Med. 185: 1043-1053.[Abstract/Free Full Text]
  17. Legge, K. L., B. Min, J. J. Bell, J. C. Caprio, L. Li, R. K. Gregg, H. Zaghouani. 2000. Coupling of peripheral tolerance to endogenous interleukin 10 promotes effective modulation of myelin-activated T cells and ameliorates experimental allergic encephalomyelitis. J. Exp. Med. 191: 2039-2052.[Abstract/Free Full Text]
  18. Legge, K. L., J. J. Bell, L. Li, R. K. Gregg, J. C. Caprio, H. Zaghouani. 2001. Multi-modal antigen specific therapy for autoimmunity. Int. Rev. Immunol. 20: 593-611.[Medline]
  19. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T cell subset inhibits antigen-specific T cells responses and prevent colitis. Nature 389: 737-742.[Medline]
  20. Wakkach, A., N. Fournier, V. Brun, J. P. Breitmayer, F. Cottrez, H. Groux. 2003. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 18: 605-617.[Medline]
  21. Waldner, H., M. J. Whitters, R. A. Sobel, M. Collins, V. K. Kuchroo. 2000. Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor. Proc. Natl. Acad. Sci. USA 97: 3412-3417.[Abstract/Free Full Text]
  22. Waldner, H., M. Collins, V. K. Kuchroo. 2004. Activation of antigen-presenting cells by microbial products breaks self tolerance and induces autoimmune disease. J. Clin. Invest. 113: 990-997.[Medline]
  23. Tuohy, V. K., Z. Lu, R. A. Sobel, R. A. Laursen, M. B. Lees. 1989. Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. J. Immunol. 142: 1523-1527.[Abstract]
  24. Greer, J. M., V. K. Kuchroo, R. A. Sobel, M. B. Lees. 1992. Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178-191) for SJL mice. J. Immunol. 149: 783-788.[Abstract]
  25. Brocke, S., K. Gijbels, M. Allegretta, I. Ferber, C. Piercy, T. Blankenstein, R. Martin, U. Utz, N. Karin, D. Mitchell, et al 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. [Published erratum in 1998 Nature 392: 630.]. Nature 379: 343-346.[Medline]
  26. Bernard, C. C. A., T. G. Johns, A. Slavin, M. Ichikawa, C. Ewing, J. Lui, J. Bettadapura. 1997. Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis. J. Mol. Med. 75: 77-88.[Medline]
  27. Min, B., K. L. Legge, C. Pack, H. Zaghouani. 1998. Neonatal exposure to a self-peptide-immunoglobulin chimera circumvents the use of adjuvant and confers resistance to autoimmune disease by a novel mechanism involving interleukin 4 lymph node deviation and interferon {gamma}-mediated splenic anergy. J. Exp. Med. 188: 2007-2017.[Abstract/Free Full Text]
  28. Pack, C. D., A. E. Cestra, M. Booki, K. L. Legge, L. Li, J. C. Caprio-Young, J. J. Bell, R. K. Gregg, H. Zaghouani. 2001. Neonatal exposure to antigen primes the immune system to develop responses in various lymphoid organs and promotes bystander regulation of diverse T cell specificities. J. Immunol. 167: 4187-4195.[Abstract/Free Full Text]
  29. Legge, K. L., R. K. Gregg, R. Maldonado-Lopez, L. Li, J. C. Caprio, M. Moser, H. Zaghouani. 2002. On the role of dendritic cells in peripheral T cell tolerance and modulation of autoimmunity. J. Exp. Med. 196: 217-227.[Abstract/Free Full Text]
  30. Li, L., H.-H. Lee, J. J. Bell, R. K. Gregg, J. S. Ellis, A. Gessner, H. Zaghouani. 2004. IL-4 utilizes an alternative receptor to drive apoptosis of Th1 cells and skews neonatal immunity towards Th2. Immunity 20: 429-440.[Medline]
  31. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336.[Medline]
  32. Lehmann, P. V., T. Forsthuber, A. Miller, E. E. Sercarz. 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358: 155-157.[Medline]
  33. Yu, M., J. M. Johnson, V. K. Tuohy. 1996. Predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease. J. Exp. Med. 183: 1777-1788.[Abstract/Free Full Text]
  34. Thornton, A. M., C. A. Piccirillo, E. M. Shevach. 2004. Activation requirement for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34: 366-376.[Medline]
  35. Chen, T. C., S. P. Cobbold, P. J. Fairchild, H. Waldman. 2004. Generation of anergic and regulatory T cells following prolonged exposure to a harmless antigen. J. Immunol. 172: 5900-5907.[Abstract/Free Full Text]
  36. Ostroukhova, M., C. Sequin-Devaux, T. B. Oriss, B. Dixon-McCarthy, L. Yang, B. T. Ameredes, T. E. Corcoran, A. Ray. 2004. Tolerance induced by inhaled antigen involves CD4+ T cells expressing membrane-bound TGB-{beta} and Foxp3. J. Clin. Invest. 114: 28-38.[Medline]
  37. Hori, S., M. Haury, A. Coutinho, J. Demengeot. 2002. Specificity requirements for selection and effector functions of CD25+CD4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice. Proc Nat Acad. Sci. USA 99: 8213-8218.[Abstract/Free Full Text]
  38. Tanchot, C., F. Vasseur, C. Pontoux, C. Garcia, A. Sarukhan. 2004. Immune regulation by self-reactive T cells is antigen specific. J. Immunol. 172: 4285-4291.[Abstract/Free Full Text]
  39. Stassen, M., H. Jonuleit, C. Muller, M. Klein, C. Richter, T. Bopp, S. Schmitt, E. Schmitt. 2004. Differential regulatory capacity of CD25+ T regulatory cells and preactivated CD25+ T regulatory cells on development, functional activation, and proliferation of Th2 cells. J. Immunol. 173: 267-274.[Abstract/Free Full Text]
  40. Tarbell, K. V., S. Yamazaki, K. Olson, P. Toy, R. M. Steinman. 2004. CD25+CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 199: 1467-1477.[Abstract/Free Full Text]
  41. Gregg, R. K., R. Jain, S. J. Schoenleber, R. Divekar, J. J. Bell, H.-H. Lee, P. Yu, H. Zaghouani. 2004. A sudden decline in active membrane-bound TGF-{beta} impairs both Treg function and protection against autoimmune diabetes. J. Immunol. 173: 7308-7316.[Abstract/Free Full Text]
  42. Gregg, R. K., J. J. Bell, H.-H. Lee, R. Jain, S. J. Schoenleber, R. Divekar, H. Zaghouani. 2005. Interlukin-10 diminishes CTLA-4 expression on islet-resident T cells and sustains their activation rather than tolerance. J. Immunol. 174: 662-670.[Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2005 174: 6559-6560. [Full Text]  



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. Ochoa-Reparaz, A. Rynda, M. A. Ascon, X. Yang, I. Kochetkova, C. Riccardi, G. Callis, T. Trunkle, and D. W. Pascual
IL-13 Production by Regulatory T Cells Protects against Experimental Autoimmune Encephalomyelitis Independently of Autoantigen
J. Immunol., July 15, 2008; 181(2): 954 - 968.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Yu, C. L. Haymaker, R. D. Divekar, J. S. Ellis, J. Hardaway, R. Jain, D. M. Tartar, C. M. Hoeman, J. A. Cascio, A. Ostermeier, et al.
Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells
J. Immunol., July 1, 2008; 181(1): 73 - 80.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Scabeni, M. Lapilla, S. Musio, B. Gallo, E. Ciusani, L. Steinman, R. Mantegazza, and R. Pedotti
CD4+CD25+ Regulatory T Cells Specific for a Thymus-Expressed Antigen Prevent the Development of Anaphylaxis to Self
J. Immunol., April 1, 2008; 180(7): 4433 - 4440.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
G. Tunheim, K. W. Schjetne, I. B. Rasmussen, L. M. Sollid, I. Sandlie, and B. Bogen
Recombinant antibodies for delivery of antigen: a single loop between {beta}-strands in the constant region can accommodate long, complex and tandem T cell epitopes
Int. Immunol., March 1, 2008; 20(3): 295 - 306.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
K. FUJIO, T. OKAMURA, A. OKAMOTO, and K. YAMAMOTO
T Cell Receptor Gene Therapy for Autoimmune Diseases
Ann. N.Y. Acad. Sci., September 1, 2007; 1110(1): 222 - 232.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
N. Kobayashi, H. Kobayashi, L. Gu, T. Malefyt, and T. J. Siahaan
Antigen-Specific Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor
J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 879 - 886.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. A. O'Connor, K. H. Malpass, and S. M. Anderton
The Inflamed Central Nervous System Drives the Activation and Rapid Proliferation of Foxp3+ Regulatory T Cells
J. Immunol., July 15, 2007; 179(2): 958 - 966.
[Abstract] [Full Text] [PDF]