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Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells¹

Ping Yu^2,*, Cara L. Haymaker^*, Rohit D. Divekar^*, Jason S. Ellis^*, John Hardaway^*, Renu Jain^*, Danielle M. Tartar^*, Christine M. Hoeman^*, Jason A. Cascio^*, Austin Ostermeier^* and Habib Zaghouani^3,*,

^* Department of Molecular Microbiology and Immunology and Department of Child Health, University of Missouri School of Medicine, Columbia, MO 65212

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.

	Introduction

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It is well documented that the thymus plays an essential role in the establishment of self tolerance (1, 2). This function involves deletion of potentially hazardous self-reactive effector T cells (central tolerance) and release of suppressive regulatory T cells (Tregs),⁴ which play a major role in peripheral tolerance of autoreactive T cells that escape thymic selection (3, 4, 5). Although the selection process of conventional T cells is well established, thymic development of Tregs remains largely undefined (6, 7). Herein, an in vivo model was developed and adapted for investigation of the role that thymic selection plays in the development of Tregs. Three reagents were used to construct this model: the SJL/J mouse, altered peptides, and immunoglobulins (Igs) carrying the altered peptides.

The SJL/J mouse expresses only the DM20 form of proteolipid protein (PLP) during fetal and neonatal life (8). DM20 is a splice variant of PLP missing the immunodominant PLP1 sequence corresponding to amino acid residues 139–151 of PLP (9). Owing to this genetic trait of the SJL/J mouse, thymic negative selection against PLP1 is defective during the fetal/neonatal period, and the mice accumulate a high frequency of PLP1-reactive T cells in the normal autoimmune repertoire (8, 10). Likewise, selection of Tregs on PLP1 should not be operative during such a period.

Igs can cross the maternal placenta and transfer from mother to fetus (10). Igs are also permissive for molecular grafting and expression of peptides within the H chain CDR-3 (11, 12, 13). Furthermore, due to efficient internalization into APCs via Fc receptors, peptide delivery by Igs enhances presentation to T cells by 100-1000-fold relative to free peptide (11). If PLP1 or PLP1-derived altered peptide ligands (APLs) are expressed on Igs, the resulting Ig-PLP1 or Ig-APL can cross the maternal placenta, undergo presentation by thymic APCs, and restore PLP1-mediated T cell selection in the SJL/J mouse.

Mutating the TCR contact residues within a peptide generates an APL that still binds to MHC molecules equally as well as the prototype peptide (14). However, stimulation of the T cells is usually reduced relative to the nominal peptide due to decrease in the affinity of the TCR to the altered peptide (15). A few APLs have been generated from PLP1 by substitution of the TCR contact residues 144 and 147 (14, 15). PLP-Y is derived from PLP1 by changing the major TCR contact residue 144W to 144Y. PLP-LR is generated by mutating aa 144W to 144L and the secondary TCR contact residue 147H to 147R. These APLs have shown a degenerate decrease in the avidity of their respective interactions with the PLP1-specific TCR, which resulted in proportional decrease in T cell stimulation in the following order: PLP1 > PLP-Y > PLP-LR (14, 15).

Herein, nucleotide sequences coding for PLP1, PLP-Y, and PLP-LR were inserted into an H chain variable region of an Ig and the mutant H chain genes were transfected into non-Ig producing SP2/0 myeloma cells along with the parental L chain to express complete Ig-PLP1, Ig-PLP-Y, and Ig-PLP-LR Ig molecules as previously described (11). The chimeras preserved the respective affinity of the peptides because Ig-PLP1 was superior to Ig-PLP-Y in stimulating the PLP1-specific TCR transgenic T cells, and Ig-PLP-LR displayed the least stimulation. When Ig-PLP1 was given to pregnant SJL/J mice on day 19 of gestation, thymic APCs from the offspring born on day 21 were able to stimulate the 5B6 TCR transgenic T cells, indicating that the chimera was able to transfer through the maternal placenta and present PLP1 peptide in fetal thymus. Moreover, for offspring born to mothers recipient of Ig-PLP1 or Ig-PLP-Y, the high-affinity ligands had increased numbers of peripheral Tregs relative to mice born to mothers not recipient of any Ig-chimera. These Tregs were suppressive and contributed to resistance against EAE. In contrast, offspring born to mothers recipient of Ig-PLP-LR, the low-affinity ligand, did not increase the number of peripheral Tregs and the mice were not protected against experimental allergic encephalomyelitis (EAE). These findings indicate that high-affinity ligands sustain thymic development of Tregs that contribute to the maintenance of peripheral tolerance.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

SJL/J (H-2^s) mice were purchased from The Jackson Laboratory and bred and maintained in our animal care facility for the duration of the experiments. RAG-2-deficient (RAG-2^–/–) SJL/J mice and 5B6 transgenic mice carrying PLP1-specific 5B6 TCR as transgene (H-2^s background) were previously described (16). 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 an encephalitogenic sequence corresponding to aa 139–151 of PLP (9). PLP-LR (HSLGKLLGRPDKF) is an altered peptide form of PLP1 in which the TCR contact residues Trp¹⁴⁴ and His¹⁴⁷ were replaced with Leu and Arg, respectively (11, 14). PLP-Y (HSLGKYLGHPDKF) is an altered peptide form of PLP1 in which the TCR contact residue Trp¹⁴⁴ was replaced with Tyr¹⁴⁴ (15). PLP1, PLP-LR, and PLP-Y peptides bind equally well to I-A^s class II molecules (14, 15).

Ig chimeras. PLP1 and PLP-LR were expressed within the heavy (H) chain variable region of the anti-arsonate Ab, 91A3, and the resulting Ig chimeras designated Ig-PLP1 and Ig-PLP-LR, respectively, were previously described (11, 12, 13). PLP-Y was expressed on 91A3 IgG2b using similar procedures as Ig-PLP1 and Ig-PLP-LR. Briefly, the diversity (D) segment within the 91A3 H chain variable region gene was deleted and replaced with a nucleotide sequence coding for PLP-Y. The chimeric 91A3V_H-PLP-Y gene was then ligated to a gene encoding a mouse 2b constant region to generate a DNA segment that encodes a full Ig H chain wherein the D segment, which is part of the CDR-3, has been replaced with nucleotides that encode PLP-Y. The chimeric H chain was then subcloned into an expression vector that harbors the gpt gene that confers resistance to mycophenolic acid. This vector was then transfected together with the pSV-neo vector harboring the parental 91A3 L chain gene into the non-Ig producing SP2/0 myeloma B cell line as described (11, 12, 13). Transfectants surviving drug selection were then assayed for production of kappa bearing IgG2b Abs by a capture ELISA, and those producing at least 2 µg/ml were selected for the study. Ig-W is the parental Ig backbone not encompassing any myelin or other peptide (11). All chimeras were grown in large-scale culture of DMEM containing 10% iron-enriched calf serum (HyClone) and were purified from culture supernatant on affinity chromatography columns made of rat anti-mouse -chain coupled to CNBr-activated Sepharose 4B (Amersham Biosciences). To avoid cross-contamination, separate columns were used to purify each chimera. Table I illustrates the Ig chimeras used in this study.

Aggregation of the Ig chimeras was done using 50%-saturated (NH₄)₂SO₄ as described previously (12, 13). Because all the Ig chimeras were derived from the same Ig backbone and thereby comprise identical IgG2b isotype, their H chain constant regions are identical and bind to Fc receptors to a similar extent.

CNS homogenate

Frozen unstripped rat brains (Pel-Freez Biologicals) were homogenized in PBS using a Waring blender and adjusted to 300 mg/ml with PBS as described previously (12, 13).

Analysis of functional avidity

To test whether PLP1 and its analogs preserve their functional avidity within the Ig context, graded amounts of Ig-PLP1, Ig-PLP-Y, Ig-PLP-LR, or the control Ig-W were incubated with splenic cells from 5B6 TCR transgenic mice containing PLP1-specific T cells as well as APCs for 3 days, and 1 µCi/well [³H]thymidine was added to the culture during the last 14.5 h. The cells were then harvested, washed three times, and incorporated [³H]thymidine was counted on a Trilux 1450 Microbeta Wallac using the Microbeta 270.004 software (Wallac). Control media without stimulation was included and used as background.

Fetal presentation of Ig-PLP chimeras

To assess for fetal presentation of the Ig chimeras, breeding females were checked for vaginal plugs daily and pregnant mice were given 300 µg of Ig-PLP1 or Ig-W i.v. at day 19 of gestation. On day 21 the newborns were sacrificed and their splenic and thymic cells were tested for stimulation of PLP1-specific 5B6 T cells without addition of exogenous Ag. Accordingly, graded numbers of thymic or splenic cells were irradiated (3000 rads) and incubated with 5 x 10⁴ 5B6 PLP1-specific T cells without the addition of exogenous Ag in a total volume of 200 µl. After 72 h, 1 µCi per well of [³H]thymidine was added during the last 15 h of incubation. The cells were then harvested and incorporated [³H]thymidine was counted as above.

Fetal exposure to Ig-PLP chimeras

Breeding females were checked for vaginal plugs daily and pregnant mice were injected i.v. with 100 µg of Ig-PLP1, Ig-PLP-Y, Ig-PLP-LR, or Ig-W on days 16, 17, and 18 of gestation. Offspring born from these mothers were used for analysis of Tregs at the neonatal stage or at the age of 6–8 wk.

Expansion and isolation of Tregs

Expansion of Tregs was done according to a previously defined regimen (17). Briefly, adult 6–8-wk-old offspring born from females recipient of Ig chimeras during pregnancy were given three injections of 300 µg aggregated (agg) Ig-PLP1 i.p. at 4-day intervals. Ten days after the last injection, the mice were sacrificed and their spleen cells were harvested for purification of Tregs. Isolation of Tregs was performed by negative selection of CD4⁺ cells and positive selection of CD4⁺CD25⁺ T cells on anti-CD25 microbeads according to Miltenyi Biotec’s instructions. The purity of cells is usually 90–95%.

Flow cytometry for analysis of Treg phenotype

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-TCR-Vβ6 (RR4–7) and PerCP-conjugated anti-CD25 (PC61) Abs at 4°C for 30 min. Subsequently, the cells were fixed and used for intracellular staining of FoxP3. 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.

Proliferation assay

Proliferation assay was used to test for in vitro suppression of CD4⁺CD25^– by CD4⁺CD25⁺ Tregs. Accordingly, 2 x 10⁵ CD4⁺CD25^– T cells were incubated with irradiated (3000 rad) SJL splenocytes as APCs, PLP1 peptide (30 µg/ml), and graded numbers of CD4⁺CD25⁺ Tregs. Two days later, 1 µCi per well of [³H]thymidine was added and the culture was continued for 15 h. The cells were then harvested and incorporated [³H]thymidine was counted.

Measurement of IL-10 by ELISA

Cytokine secretion by Tregs was performed by incubating 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 a standard protocol provided by 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 Tregs rather than by APCs.

Induction of EAE

Active EAE. This was done as described previously (12, 13). 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. 75 ng purified Bordetella pertussis toxin (List Biological Laboratories). A second injection of 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.

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

Suppression of EAE with Tregs

For suppression of active EAE, SJL/J mice were given 1 x 10⁶ purified CD4⁺CD25⁺ T cells, induced for EAE the next day and monitored for clinical signs of disease for 30–40 days.

For suppression of passive EAE, RAG-2^–/– mice were given 0.5 x 10⁶ CD4⁺CD25⁺ T cells along with 1 x 10⁶ naive 5B6 TCR Tg T cells and the animals were monitored for clinical signs of EAE.

Immunization with PLP1 peptides

Six- to 8-wk-old SJL/J offspring mice that were exposed to Ag during fetal life were immunized by s.c. injection in the footpads and at the base of the limbs of a 200 µl CFA/PBS (v/v) solution containing 100 µg of PLP1 peptide, and cytokine production by splenic cells was analyzed 10 days after immunization.

Depletion of CD4⁺CD25⁺ T cells in vivo

Depletion of CD4⁺CD25⁺ T cells was done as described previously (17). Briefly, the mice were given two i.p. injections of anti-CD25 Ab (PC61) 4 and 2 days before the induction of disease with 100 µg PLP1 peptide in CFA. 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.

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

 
Altered peptides expressed on Igs preserve their functional avidity for stimulation of naive T cells as well as expansion of Tregs

PLP1 peptide, the prototype epitope for the 5B6 TCR transgenic T cells, and the PLP1-derived altered peptides PLP-Y and PLP-LR were expressed on Ig, and the resulting Ig-PLP1, Ig-PLP-Y, and Ig-PLP-LR were tested for stimulation of 5B6 cells to assess whether grafting onto Ig affected the functional avidity of the peptides. The TCR affinity for the peptides was previously determined and PLP1 had the highest affinity, while PLP-Y was median and PLP-LR was the lowest (14, 15). T cell stimulation by Ag, which represents the functional avidity of TCR-ligand interactions, usually reaches an optimal level with low concentrations for high-affinity Ags, but requires much higher amounts for low-affinity Ags. In the context of Ig, the prototype peptide PLP1, which has the highest affinity to PLP-specific TCR transgenic T cells (16), also displayed the strongest stimulatory function. Indeed, Fig. 1 shows that Ig-PLP1 induced much higher proliferation of 5B6 T cells than did any of the other constructs. Ig-PLP-Y was moderately stimulatory while Ig-PLP-LR was almost nonstimulatory except at the highest concentration used, where some significant proliferation was observed relative to the control Ig-W, the backbone Ig without any PLP peptide. In fact, higher concentration of Ig-PLP-LR led to similar low but consistent proliferation (not shown). Overall, expression of peptides on Igs did not alter the functional avidity of the peptide and the stimulation function ranks as follows: Ig-PLP1 > Ig-PLP-Y > Ig-PLP-LR.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Expression of altered peptides on Igs preserves functional avidity. Splenocytes (5 x 105 per well) from 5B6 TCR transgenic mice containing both PLP1-specific T cells and APCs were incubated with graded amounts of Ig chimeras for 3 days. Subsequently, [3H]thymidine 1 µCi/well was added and the culture continued for an additional 14.5 h. The cells were then harvested and the incorporated [3H]thymidine was counted on a Trilux 1450 Microbeta counter. Each point represents the mean of triplicate wells. The results are representative of three independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Aggregated Ig-PLP1 is more effective than Ig-PLP-Y or Ig-PLP-LR for expansion of PLP1-specific Tregs. Naive PLP1-specific 5B6 TCR Tg CD4+ T cells were injected into adult SJL/J mice (5 x 106 cells/mouse) i.v. through the tail vein and the recipients were given an i.p. injection of 300 µg of agg Ig chimeras in saline on days 4, 8, and 12 posttransfer. Ten days later, the splenic CD4+ T cells were isolated by positive selection on anti-CD4 Ab coupled to microbeads (Miltenyi Biotec). The CD4 T cells (2 x 106 cells/sample) were analyzed for cell surface expression of TCRVβ6 (left panels), coexpression of CD25 (middle panels), or CD25 and FoxP3 (right panels) by flow cytometry. The number in the box indicates the percentage of cells expressing the corresponding marker among total CD4 cells. The results are representative of five different experiments.

Because the study presented herein investigates Treg selection in vivo rather than in fetal thymic organ culture in vitro, we had to ensure that the Ig chimeras cross the maternal placenta, transfer from mother to fetus, reach the fetal thymus, and are presented by thymic APCs. Accordingly, pregnant mice were injected i.v. with soluble (nonaggregated) Ig-PLP1 or the control Ig-W on day 19 of gestation, and thymic and splenic cells from neonates born on day 21 were assayed for stimulation of PLP1-specific TCR Tg 5B6 T cells without addition of exogenous Ag. The results in Fig. 3 show that both thymic and splenic APCs from mice born to Ig-PLP1 recipient mothers stimulate the proliferation of 5B6 T cells, whereas cells from mice born to Ig-W recipient mothers did not. These data indicate that the transferred Ig-PLP1 was taken up by APCs and that I-A^s-PLP1 complexes were generated in both the central and peripheral fetal lymphoid organs and stimulated 5B6 T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Ig-PLP1 transfers from mother to fetus and drives peptide presentation by fetal thymic and splenic APCs. Pregnant SJL/J female mice were given 300 µg of Ig-PLP1 or control Ig-W on day 19 of gestation, and graded numbers of thymic (A) and splenic (B) cells of the offspring born on day 21 were irradiated and assayed for activation of PLP1-specific TCR transgenic 5B6 CD4+ T cells (5 x 104 cell per well) without adding exogenous Ags. Proliferation was measured by [3H]thymidine incorporation. Each point represents the mean ± SD of triplicates.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Fetal exposure to high-avidity chimeras magnify Treg generation. Pregnant female SJL/J mice were given Ig-PLP1, Ig-PLP-Y, or Ig-PLP-LR on days 16, 17, and 18 of gestation, and the offspring born on day 21 were allowed to grow into adults. At 6 wk of age the offspring were given three injections of agg Ig-PLP1 (300 µg/injection) 4 days apart, and 10 days later the splenic CD4+ T cells were purified and stained for surface CD25 and CTLA4, as well as for intracellular FoxP3, as described in Fig. 3. One group of mice that had not been exposed to any Ag during fetal life (NIL), but that received agg Ig-PLP1 for Treg expansion, was included for control purposes. Cells were gated on live cells and analyzed for CD25, CTLA4, and FoxP3 expression. The numbers in the upper right corner indicate the percentage of cells expressing the specific marker among total gated CD4+ T cells. The results are representative of three different experiments.

Previously, we have shown that fetal exposure to Ig-PLP1 leads to deletion of pathogenic T cells and confers resistance to EAE (8, 10). Because PLP-Y has lower affinity than PLP1 and does not delete CD4⁺CD25^– pathogenic cells (data not shown), we chose to use Tregs selected on Ig-PLP-Y to test for suppression of proliferation of CD4⁺CD25^– pathogenic counterparts that developed under the same fetal exposure and for protection of the offspring against EAE. Accordingly, CD4⁺CD25⁺ cells from offspring that were fetally exposed to Ig-PLP-Y were purified and tested for suppression of Ag-induced proliferation of CD4⁺CD25^– counterparts purified from the same animals. As indicated in Fig. 5, CD4⁺CD25^– T cells proliferate significantly when activated with soluble anti-CD3 Ab. However, when CD4⁺CD25⁺ T cells were added to the culture the proliferation was significantly reduced (Fig. 5A). The suppression is comparable to results obtained with natural Tregs purified from naive SJL/J mice that were not subject to fetal exposure to Ig-PLP-Y or expansion by agg Ig-PLP-1 and was very effective, as suppression remains significant even at a ratio of 1 CD4⁺CD25⁺ to 4 CD4⁺CD25^– T cells. Also, the cells produced significant amounts of IL-10 relative to the CD4⁺CD25^– counterparts when they were stimulated with plate-bound anti-CD3 Ab (Fig. 5B). Stimulation with rat IgG isotype control did not lead to increased IL-10 production (data not shown). Taken together, these results indicated that CD4⁺CD25⁺ T cells that were selected on Ig-PLP-Y are suppressive to the same extent as natural Tregs.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Ig-PLP-Y-selected CD4+CD25+ T cells suppress proliferation of their CD4+CD25– counterparts. SJL/J offspring that had been fetally exposed to Ig-PLP-Y were given agg Ig-PLP1 at the age of 6 wk, and the expanded CD4+CD25+ T cells were purified and tested for suppression of their CD4+CD25– counterparts (A) and IL-10 production (B). The cells were purified 10 days after the last injection of agg Ig-PLP1, and separation was done by MACS using a CD4 T cell isolation kit and anti-CD25 microbeads according to Miltenyi Biotec’s instructions. For measurement of suppression CD4+CD25+ T cells (2 x 105 T cells per well) were incubated with graded numbers of CD4+CD25– T cells and 4 x 105 SJL-irradiated (3000 rad) splenocytes as APCs. Soluble anti-CD3 Ab (4 µg/ml) was then added and the culture was continued for 48 h. Subsequently, [3H]thymidine was added (1 µCi/well) and proliferation was measured 14.5 h later. CD4+CD25+ T cells alone without effectors (1:0, filled bars) and CD4+CD25– effectors without Tregs (0:1, open bars) were included to serve as controls. CD4+CD25+ T cells from naive SJL/J mice that were not subject to fetal selection and expansion regimens (Natural) were included for comparison with cells that were fetally selected with Ig-PLP-Y and expanded with agg Ig-PLP-1. Each bar represents the mean ± SD of triplicates. For measurement of IL-10 (B), selected and natural CD4+CD25+ (2 x 105 cells per well) were incubated on plate-bound anti-CD3 Ab (5 µg/ml) for 48 h. IL-10 production was detected by ELISA. CD4+CD25– T cell counterparts (2 x 105 cells per well) were included to serve as control. Incubation on plate-bound hamster IgG was performed as isotype control and no measurable IL-10 was detected. Each bar represents the mean ± SD of triplicates. The results are representative of three experiments.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Ig-PLP-Y selected Treg cells ameliorate both passive and active EAE. Six-week-old SJL/J mice (A and B) were given natural () or Ig-PLP-Y-selected () Tregs (1 x 106 cells per mouse) and 1 day later induced for EAE with PLP1 peptide (A) or CNS homogenate (B). The mice were monitored for signs of paralysis daily for 40 days. A group of mice that did not receive any T cell transfer (NIL) was included as control. For passive EAE (C and D), RAG-2–/– SJL mice were given i.v. 1 x 106 naive 5B6 TCR Tg CD4+ T cells alone (5B6) to induce the disease (17 ). To test for suppression, the mice received along with 5B6 TCR Tg CD4+ T cells 0.5 x 106 natural (5B6 + Natural) or Ig-PLP-Y-selected (5B6 + Ig-PLP-Y) Tregs. The mice were monitored daily for signs of paralysis for 30 days. Each point represents the mean clinical score of five to six mice.

Fetal exposure to high-affinity ligands confers resistance against EAE in a Treg-dependent fashion

Fetal exposure to high-, but not low-, affinity ligands seeded the periphery with Tregs that were suppressive against effector T cells both in vitro and in vivo (Figs. 5 and 6). Herein we tested whether selected Tregs protect mice recipients of the fetal regimen against active EAE. Accordingly, pregnant SJL/J mothers were injected with Ig-PLP1, Ig-PLP-Y, or Ig-PLP-LR at days 16, 17, and 18 of gestation, and the offspring were allowed to grow into adults and induced for EAE with PLP1 peptide in CFA. Fig. 7 shows that fetal exposure to Ig-PLP1 resulted in only mild EAE disease relative to SJL/J mice not recipient of any fetal treatment (NIL group). Indeed, the mean maximal disease severity score was 2.8 ± 0.5 in the NIL group while the Ig-PLP1 mice had a mean maximal disease severity score of 0.8 ± 0.5 (Fig. 7A). Because Ig-PLP1 can delete effector T cells, the resistance against EAE may be the result of both elimination of effector T cells and increase in Treg development. The mice recipient of Ig-PLP-Y, the midrange affinity chimera, also decreased the severity of EAE, and the mean maximal disease score was reduced from 2.8 ± 0.5 in the NIL group to 1.8 ± 0.5 in Ig-PLP-Y mice (Fig. 7B). Note, however, that reduction in disease severity was less prominent than for Ig-PLP1 (mean maximal disease score of 1.8 for Ig-PLP-Y vs 0.8 for Ig-PLP1), and this was likely due to ineffective deletion of effector T cells by fetal exposure to Ig-PLP-Y. In contrast, mice born to mothers that received the low-affinity Ig-PLP-LR ligand developed nearly the same severity of EAE as did the NIL mice, possibly reflecting absence of deletion of effector T cells and lack of increase in Treg development (Fig. 7C). These results indicate that fetal exposure to high-affinity ligands plays a critical role in peripheral tolerance and suppression of EAE. If the suppression of EAE was due to Tregs, the splenic cells should produce IL-10 upon stimulation with PLP1 peptide, and depletion of these cells before induction of EAE should restore the disease. To test these premises, mice born to mothers that received Ig-PLP1, Ig-PLP-Y, or Ig-PLP-LR were allowed to grow into adults and injected with PLP1 peptide in CFA. Ten days later, splenocytes were assessed for IL-10 production upon stimulation with PLP1 peptide. The results show that fetal presentation of Ig-PLP1 (Fig. 7D) or Ig-PLP-Y (Fig. 7E) dramatically increased IL-10 secretion compared with offspring without any fetal exposure but that were recipients of PLP1 peptide/CFA immunization as adults (NIL group). In contrast, mice born to mothers that received Ig-PLP-LR instead of Ig-PLP1 or Ig-PLP-Y had lower levels of IL-10, which were similar to the NIL group (Fig. 7F). Production of IL-10 is Ag-specific because stimulation with the control PLP2 peptide had minimal levels in the Ig-PLP1, Ig-PLP-Y, and Ig-PLP-LR groups. Given that fetally selected and peripherally expanded Tregs produce IL-10 (Fig. 5), it is likely that the IL-10 found in the spleen of mice fetally exposed to Ig-PLP1 or Ig-PLP-Y and resistant to PLP1 EAE emanates from Tregs.

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Offspring fetally exposed to high-avidity peptides resist EAE induction and produce IL-10 cytokine. Six-week-old SJL/J offspring fetally exposed to Ig-PLP1 (A), Ig-PLP-Y (B), or Ig-PLP-LR (C) were induced for EAE with 100 µg PLP1 peptide in CFA as described in Materials and Methods and monitored daily for signs of paralysis for 30 days. A group of mice that was not exposed to any peptide during fetal life (NIL) was included for control purposes. Each point represents the mean clinical score of five to six mice. D–F, IL-10 production by splenic cells from adult SJL/J offspring fetally exposed to Ig-PLP1 (D), Ig-PLP-Y (E), or Ig-PLP-LR (F). The mice were immunized with PLP1 peptide in CFA as in A–C except that not pertussis toxin was administered to allow for T cell accumulation in the spleen. Ten days later, the splenic cells (1 x 106 cells/well) were stimulated in vitro with 20 µg/ml PLP1 or the negative control PLP2 peptide, and IL-10 secretion was measured by ELISA. A group of mice that were not fetally exposed to any PLP peptide but immunized with PLP1 peptide (NIL) was included for control purpose. Each bar represents the mean ± SD of three to five individually tested mice.

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Fetal exposure to Ig-PLP-Y facilitates Treg selection but does not inactivate pathogenic T cells. SJL/J offspring fetally exposed to Ig-PLP-Y were given two injections (1 mg per mouse per injection) of either of anti-CD25 Ab (PC61) () or Rat- IgG () on day –4 and –2 before induction of EAE with 100 µg PLP1 peptide in CFA. The animals were then monitored for clinical signs of EAE daily for 30 days. SJL/J offspring that were not fetally exposed to any PLP peptide but received anti-CD25 Ab () were used as control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The results presented herein indicate that fetal exposure to high-affinity TCR ligand promotes development of Tregs while exposure to low-affinity TCR ligand does not. The interpretation put forth for this observation states that Tregs likely require a TCR ligand avidity higher than conventional T cells for positive selection, as both Ig-PLP1 and Ig-PLP-Y restored selection in SJL/J mice and supported generation of Tregs, while the low-affinity ligand, which likely supports positive selection of conventional T cells, had no influence in thymic generation of Tregs. The significance of this opposite function of peptide avidity in effector T cells vs Treg development is that high-affinity peptides that usually support encephalitogenicity would engender deletion of effector T cells but increase development of Tregs to better maintain tolerance. Peptides or APLs with lower affinity, however, would not expand Tregs, as their effector T cells would not be highly self-reactive or pathogenic.

	Acknowledgments

 
We thank J. Jeremiah Bell for technical suggestions.

	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 2RO1 NS37406, RO1 NS057194, and RO1 AI48541 (to H.Z.) from the National Institutes of Health. J.S.E. and C.M.H. were supported by the predoctoral training grant (T32 GM08396) from National Institute of General Medical Sciences. D.M.T., J.A.C., and J.C.H. were supported by a life science fellowship from the University of Missouri.

² Current address: National Cancer Institute, Metabolism Branch, Bethesda, MD 20892.

³ 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

⁴ Abbreviations used in this paper: agg, aggregated; APL, altered peptide ligand; EAE, experimental allergic encephalomyelitis; PLP, proteolipid protein; Treg, regulatory T cell; Tg, transgenic.

Received for publication January 10, 2008. Accepted for publication April 21, 2008.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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