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TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells¹

Jie Dai^*, Bei Liu^*, Soo Mun Ngoi^*, Shaoli Sun, Anthony T. Vella^* and Zihai Li^2,*

^* Department of Immunology, University of Connecticut School of Medicine, Farmington, CT 06030; and Dianon Systems, Stratford, CT 06615

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
As one of the main mediators of the endoplasmic reticulum unfolded protein response, heat shock protein gp96 is also an obligate chaperone for multiple TLRs including TLR4. We demonstrated recently that enforced cell surface expression of gp96 in a transgenic (Tg) mouse (96tm-Tg) conferred hyperresponsiveness to LPS and induced TLR4-dependent lupus-like autoimmune diseases. In this study, we investigated the function of CD4⁺CD25⁺ Foxp3⁺ regulatory T cells (T_reg) in these mice in light of the important roles of T_reg in the maintenance of peripheral tolerance against self-Ag as well as the increasing appreciation of TLR signaling on the regulation of T_reg. We found that the development of T_reg was not impaired in 96tm-Tg mice. Contrary to the prediction of dampened T_reg activity, we discovered that the suppressive functions of T_reg were increased in 96tm-Tg mice. Inactivation of T_reg during the neonatal stage of life exacerbated not only organ-specific diseases but also systemic autoimmune diseases. By crossing 96tm-Tg mice into the TLR4 null background, we demonstrated the critical roles of TLR4 in the amplification of T_reg suppressive function. These findings illustrate that gp96 plays dual roles in regulating immune responses by augmenting proinflammatory responses and inducing T_reg function, both of which are dependent on its ability to chaperone TLR4. Our study provides strong support to the notion of compensatory T_reg activation by TLR ligation to dampen inflammation and autoimmune diseases.

	Introduction

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It has been shown that CD4⁺CD25⁺ regulatory T cells (T_reg)³ play important roles in peripheral T cell tolerance by suppressing self-reactive immune responses, regulating antitumor immunity, and fine tuning inflammatory responses to pathogens (1, 2). Much progress has been made regarding the ontogeny and phenotype of T_reg. Foxp3, a member of the forkhead/winged-helix family of transcriptional regulators, is uniquely expressed by the T_reg and is the master developmental factor for the generation of T_reg (3) through cooperation with NFAT (4). The NFAT connection explains the need for both IL-2 (5) and CD28 signaling (6) in the generation of functional T_reg.

The mechanism by which T_reg is functionally regulated remains unclear. T_reg express variable levels of CTLA-4, glucocorticoid-induced TNFR-related gene product (GITR), TGF-, IL-10, and other molecules (1). In vitro, the suppressive functions of T_reg are dependent on cell-cell contact between T_reg and CD4⁺CD25^– effector T cells (T_eff) and independent of soluble factors, such as IL-10 and TGF- (1). However, TGF- and IL-10 are required for the inhibitory function of T_reg in vivo (7). The roles of CTLA-4 and GITR have also been implicated as mediators of T_reg function (7, 8). Recently, it was found that TGF- induces the generation of Foxp3⁺ T_reg, which were completely inhibited by a proinflammatory cytokine IL-6 (9).

TLRs are important pattern recognition receptors for host to recognize and respond to pathogen-associated molecular patterns such as LPS (10). The folding and surface expression of TLR1, TLR2, and TLR4 are dependent on an endoplasmic reticulum chaperone gp96 (11). TLR ligation is generally thought to suppress T_reg function (12). Two categories of TLR activity that down-regulate the function of T_reg have been discovered: suppression of T_reg by direct ligation of TLR8 (13) and TLR2 (14, 15) on T_reg; and blunting T_reg function in a TLR4-dependent fashion by rendering T_eff less responsive to T_reg (16). In this regard, suppression of T_reg was believed to be essential for the initiation of the adaptive immune response.

Paradoxically, ligation of TLR4 (17, 18) and TLR5 (19) on T_reg was recently reported to have an opposite enhancing effect on the function of T_reg. In addition, antagonizing MD1 was also found to decrease the threshold of LPS activation of dendritic cells (DC) and to enhance the induction of T_reg (20). The T_reg-amplifying effect of TLR activation in this situation could potentially play critical roles in curtailing a late-phase immune response when an infection is contained and thus provide a feedback inhibition mechanism against TLR-dependent inflammatory process. However, direct effect of TLR4 ligation on T_reg has been challenged (14, 19, 21, 22, 23). More importantly, it is unclear whether TLR4 amplification in vivo could lead to enhanced T_reg function.

We previously reported that enforced cell surface expression of gp96 (96tm) in an otherwise non-autoimmune prone C57BL/6 mice induces systemic lupus-like autoimmune diseases (24). Furthermore, we found that 96tm expression confers hyperresponsiveness to LPS and the spectrum of autoimmunity in 96tm-transgenic (Tg) mice is completely abrogated in the absence of TLR4 (25). In this study, we addressed two interrelated questions. First, what is the impact of TLR4 hyperfunction on the function of T_reg in vivo? Second, to what extent does T_reg functional alteration, if any, contribute to the development of systemic autoimmune diseases in 96tm-Tg mice? We provide in vivo and in vitro evidence that surface expression of gp96 enhanced suppressive T_reg function, which attenuated autoimmunity as inactivation of T_reg during neonatal stage of life exacerbated autoimmune diseases in 96tm-Tg mice. More importantly, we discovered that gp96-mediated enhancement of T_reg function was dependent on TLR4. Our study is the first example of curtailing TLR4 hyperactivation via T_reg on the organismal level to prevent systemic autoimmune diseases, thus providing strong support to the notion of compensatory T_reg activation by TLR ligation to dampen inflammation.

	Materials and Methods

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Mice

Membrane-bound gp96 Tg (96tm-Tg) mice were generated as previously described (24). C57BL/6 mice were purchased from The Jackson Laboratory. TLR4^–/– mice were provided by Dr. R. Medzhitov (Yale University, New Haven, CT). Mice were maintained by the Center for Laboratory Animal Care of the University of Connecticut Health Center (Farmington, CT) according to the established guidelines (Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources Commission on Life Sciences).

Abs and other reagents

All Abs used for flow cytometry were from BD Pharmingen or eBioscience, unless otherwise stated. Anti-CD25 Ab (clone PC61) was obtained from American Type Culture Collection. FITC-labeled anti-CD25 Ab (clone 7D4) was used to confirm the inactivation efficiency of CD4⁺CD25⁺ T cells.

Flow cytometry

Briefly, cells were washed with staining buffer (cold PBS plus 2% heat-inactivated FCS), blocked on ice with a purified anti-FcRII/RIII Ab (clone 93; eBioscience) for 10 min, followed by staining with various fluorochrome-conjugated Ab against interested surface markers. After staining, cells were then washed and analyzed on FACSCalibur (BD Biosciences). Dead cells were gated out by propidium iodide exclusion. For intracellular staining, cell surface markers were first stained as described, followed by fixation in 2% formaldehyde buffered with PBS, washing and permeabilization with 0.25% saponin in the staining buffer. Further staining was done using fluorochrome-conjugated Ab against respective intracellular proteins. In some experiments, cells were activated in vitro with 50 ng/ml PMA and 1 µg/ml ionomycin for 5 h in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich).

In vitro suppression assay

CD4⁺CD25⁺ T_reg and CD4⁺CD25^– T_eff were purified using the Regulatory T Cell Purification kit (Miltenyi Biotec) according to the manufacturer’s protocol. CD4-depleted splenocytes were irradiated (3000 rad) and used as APCs. In 96-well round-bottom plates, each well contained 5 x 10⁴ T_eff, 5 x 10⁴ APCs, titrated number of T_reg, in the presence or absence of soluble anti-CD3 Ab (2.5 µg/ml). Cells were cultured at 37°C for 72 h. During the last 6 h, cells were pulsed with 1 µCi of [³H]thymidine, harvested by an automated 96-well plate harvester, and the cpm were determined by an automated liquid scintillation counter (Microplate Scintillation and Luminescence Counter; Packard Instrument). The suppression index was calculated as [CPM_{(Teff without Treg)} – CPM_{(Teff with Treg)}]/CPM_{(Teff without Treg)} x 100%.

Determination of staphylococcal enterotoxin A (SEA)-specific CD4⁺ T cell response

Mice were treated with either control IgG or PC61 (anti-CD25) 3 days before immunization. Inactivation efficacy was verified by flow cytometry using anti-CD25 (7D4) Ab recognizing a different epitope from PC61. One day after i.p. injection of 1 µg of SEA, 25 µg of LPS (from Salmonella typhimurium; Sigma-Aldrich) was administered at the same route. Mice were bled for the kinetic analysis of SEA-specific V3⁺ CD4⁺ T cells as well as control V14⁺ CD4⁺ T cells in peripheral blood. At day 16, mice were sacrificed, and the percentage and the absolute number of SEA-selective and nonselective CD4⁺ T cells was determined from various organs.

Inactivation of T_reg during the neonatal stage of life

Newborn mice were treated with PC61 Abs (500 µg) or control rat IgG by i.p. injection every 4 days for four doses, starting before 9 days of age until day 21. Antinuclear Ab (ANA) was tested using sera obtained at weeks 8, 10, and 12. Mice were sacrificed 5 mo later, for evaluation of disease and serum level of cytokines.

ELISA

ELISA was used for the quantification of serum cytokines (BD Pharmingen) according to the manufacturer’s protocol.

Histology

Various organs were fixed in 4% formaldehyde buffered with PBS. The 5-µm sections were cut using Shandon Cryotome E (Thermo Electron), stained with H&E by standard methods, and examined by a light microscopy. The evaluation was performed by single blind testing, and the severity of the gastritis was scored as follows: 0 (no inflammation), 1 (mild lymphocytic infiltration), 2 (moderate lymphocytic infiltration), or 3 (severe lymphocytic and neutrophil infiltration).

Immunofluorescence detection of glomerulonephritis and clinical evaluation of the severity of kidney disease

Samples of 5-µm cryosections of kidneys were incubated with blocking reagent (Vector Laboratories), stained with biotinylated anti-mouse IgG followed by FITC-conjugated streptavidin. Each sample was examined under a fluorescent microscope. Every glomerulus was assigned a number for its fluorescence intensity as follows: 0 (negative), 1 (slight staining), 2 (moderate staining), or 3 (bright staining). Based on an average of 83.5 ± 20.3 glomeruli evaluated per mouse (mean ± SD, obtained from a total of 40 mice), the mean fluorescence intensity was calculated as the index for the disease severity.

Detection of ANA

HEp-2-coated slides (INOVA Diagnostics) were incubated with diluted sera, followed by FITC-conjugated goat anti-mouse Ig, and examined by a fluorescence microscopy. The fluorescence staining intensity was graded as 0 (negative), 1 (slight staining), 2 (moderate staining), and 3 (bright staining). The mean fluorescence intensity was calculated.

RT-PCR

Total RNA was extracted from purified wild-type (WT) or 96tm-Tg T_reg using a NucleoSpin RNA II kit (with DNase I on-column digestion), according to the manufacturer’s protocol (Clontech Laboratories). Total RNA was reverse transcribed using Superscript II and oligo(dT)_12–18 primer (Invitrogen Life Technologies) per the manufacturer’s protocol. The resulting cDNA obtained from 200 ng of total RNA was amplified for genes of interests for 35 cycles, using the following primers: gp96 (forward) 5'-ccttgacattggttattgacg-3', (reverse) 5'-ccttctcggcttttacccagg-3'; 96tm (forward) 5'-acaccaaggcgtatggagat-3', (reverse) 5'-gatgatggtgagcaccaccag-3'; and -actin (forward) 5'-tcagaaggactcctatgtgg-3', (reverse) 5'-tctctttgatgacacgcacg-3'.

Statistical analysis

Student’s t test or Mann-Whitney U test was used for the statistical analysis by Prism software (GraphPad software). General linear model univariate and multivariate analysis were performed using SPSS 12.0 software. Values of p < 0.05 were considered statistically significantly different.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Normal development of 96tm-Tg Foxp3⁺ T_reg

Both male and female 96tm-Tg mice developed the serological/histological evidence of autoimmunity around 4 mo. To understand the possible involvement of T_reg in the pathogenesis of autoimmunity, we first analyzed T_reg in 5-mo-old 96tm-Tg mice and age- and gender-matched WT mice to determine whether there was any developmental alteration of T_reg in 96tm-Tg mice. By intracellular staining for Foxp3 protein, the best marker for CD4⁺CD25⁺ natural T_reg, we determined the percentage and absolute number of T_reg in the spleen and thymus of both WT and 96tm-Tg mice (3). We found that within the CD4⁺CD8^– single-positive population, 7% cells expressed surface CD25 (Fig. 1A) and 2–3% were CD4⁺ Foxp3⁺ T cells in both Tg and WT mice (Fig. 1B). No Foxp3 positivity was found in CD4^– populations. Similar to thymocyte T_reg distribution, splenic CD4⁺CD25⁺ populations were comparable between WT and Tg mice, either in percentage (Fig. 1C) or in absolute number (data not shown). Most of these cells were phenotypically T_reg as over 80% of them were also positive for Foxp3 (data not shown). Fewer than 5% of CD4⁺CD25^– splenocytes were Foxp3⁺. Tg CD4⁺CD25⁺ T cells expressed similar levels of GITR on their cell surface (Fig. 1C), although no surface CTLA-4 or TGF- was detectable on unstimulated splenic CD4⁺CD25⁺ T cells from either WT or 96tm-Tg mice (data not shown). The total number of CD4⁺ Foxp3⁺ cells in the spleen was the same between WT and Tg mice, although the percentage of CD4⁺ Foxp3⁺ T cells in the spleen of 96tm-Tg mice was slightly reduced (Fig. 1D).

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Phenotypic comparison between WT and 96tm-Tg Treg. A and B, Thymocytes from 5-mo-old WT and 96tm-Tg mice were subjected to flow cytometric analysis. CD4+CD8– single-positive populations were gated on. A, Surface staining of CD25 marker gated on propidium iodide (PI–) populations. B, Intracellular staining for Foxp3. C and D, Flow cytometric analysis of splenocytes from 5-mo-old mice. C, Surface staining of CD25 or GITR gated on PI– populations. D, Intracellular staining of Foxp3. IgG2a and IgG2b represent isotype controls for Ab against Foxp3 and GITR respectively. Data of one representative mouse from more than six mice in several independent experiments is shown.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Cytokine profile of WT and 96tm-Tg splenocytes after polyclonal activation in vitro. Splenocytes from 5-mo-old WT and 96tm-Tg mice were incubated with PMA/ionomycin for 5 h in the presence of brefeldin A. Intracellular staining of individual cytokine and respective isotype controls were performed after surface staining of CD4 and CD25. Data of one representative mouse from three mice is shown for all, except that the staining for IL-10, and its isotype control represents analysis of 12 mice.

Next, we performed functional analysis of T_reg from WT and Tg mice using a standard in vitro suppression assay to determine whether 96tm T_reg retains suppressive function. CD4⁺CD25⁺ T_reg were purified from the WT and Tg splenocytes by sequential negative (CD4^– lineage mixture) and positive (CD25⁺) selections, which were then cocultivated with CD4⁺CD25^– T_eff in the presence of splenic APCs (CD4⁺ T cell-depleted populations) and soluble anti-CD3 Ab for 3 days. T cell proliferation was indexed by [³H]thymidine incorporation during the last 6 h of culture. In the absence of T_reg, both WT and Tg T_eff proliferated robustly (Fig. 3, A and B). In contrast, neither WT nor Tg T_reg divided, which is in agreement with the anergic property of T_reg in vitro (1). The proliferation of T_eff was greatly reduced in the presence of either WT or Tg T_reg at 1:10 or 1:1 ratio (Fig. 3, A and B). The suppressive function of 96tm-Tg T_reg was significantly more pronounced than that of WT T_reg on the per cell basis, regardless whether T_eff and APCs were of WT or Tg origin in these assays (Fig. 3).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Increased suppressive function of CD4+CD25+ splenic Treg in 96tm-Tg mice in vitro. CD4+CD25+ Treg were purified from WT or 96tm-Tg splenocytes, respectively. A total of 5 x 104 CD4+CD25– Teff were cocultured in the presence of soluble anti-CD3 mAb for 72 h with medium, 0.5 x 104 (1:10) or 5 x 104 (1:1) Treg. [3H]Thymidine was added during the last 6 h. A, A total of 5 x 104 CD4-depleted splenocytes from WT mice were used as APCs. B, A total of 5 x 104 CD4-depleted splenocytes from 96tm-Tg mice served as APCs. At least three independent experiments were performed with the similar findings. Error bars represent SD. C, Comparisons of suppressive function between WT and 96tm-Tg Treg from young and old mice. Proliferation obtained in the absence of anti-CD3 mAb was generally under 200 cpm (data not shown). Symbols represent results from one individual experiment with Treg purified from the pooled splenocytes of two to three mice. The bars represent the mean values.

Regulatory effects of 96tm-Tg T_reg on T_eff in vivo

If T_reg is hyperfunctional in 96tm-Tg mice in vivo, inactivation of T_reg might lead to more robust immune responses. To test this possibility, we kinetically analyzed the expansion of endogenous SEA-specific CD4⁺ T cells after immunization with SEA, using low-dose LPS (25 µg) as an adjuvant (28, 29), in the presence or absence of functional T_reg. We injected mice with the mAb PC61, which recognizes the -chain of the IL-2R, CD25; this Ab is known to specifically deplete T_reg (30, 31, 32, 33). The report of inactivation rather than depletion of the CD4⁺CD25⁺ T_reg in vivo by anti-CD25 Ab (34) was most likely related to distinct Ag specificity of anti-CD25 Ab (32, 33). PC61 was i.p. administered 3 days before SEA immunization. SEA-specific V3⁺ CD4⁺ T cells in the peripheral blood were analyzed by flow cytometry (Fig. 4A). The kinetics of the expansion of WT and Tg SEA-specific V3⁺ CD4⁺ T cells mirrored each other well when T_reg was unmanipulated. In the absence of functional T_reg, the Ag-specific V3⁺ CD4⁺ T cells expanded much more in both WT and Tg mice, but the magnitude of expansion of V3⁺ CD4⁺ T cells from 96tm-Tg mice were substantially greater than WT V3⁺ CD4⁺ T cells (p = 0.049 by univariate regression analysis) (Fig. 4A). Furthermore, the increased percentages of Ag-specific V3⁺ CD4⁺ T cells were also found in other lymphoid compartments, such as spleen (p = 0.005 by multivariate analysis) or inguinal, auxiliary, and cervical lymph nodes (p = 0.006) and mesenteric lymph nodes (p = 0.008) (Fig. 4C). In contrast, non-SEA-specific V14⁺ CD4⁺ T cells in peripheral blood as well as lymph nodes remained constant in either WT or Tg mice (Fig. 4, B and D). Our data demonstrated that Tg T_reg were not only hyperfunctional in vitro, but also exerted significantly more suppressive regulatory functions on the expansion of CD4⁺ T_eff in vivo.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Increased expansion of Ag-specific CD4+ T cells after in vivo inactivation of Treg. Mice were injected i.p. with PC61 or control IgG before immunization with SEA plus LPS. PBL were subject to flow cytometry after staining with CD4 and TCR V3 (A) or with CD4 and TCR V14 (B) at different time points. *, p < 0.05. Splenocytes, mesenteric lymph nodes (MLN), and pooled cervical, inguinal and auxiliary lymph nodes (sLN) were also analyzed on day 16 to determine the percentage of V3 (C) or V14 (D) CD4+ T cells. **, p < 0.01. Two independent experiments with three to four mice in each group were performed with similar results. The error bars represent the SEM of at least three mice per group.

Having observed the increased suppressive function of Tg T_reg in vitro and their regulatory roles in vivo, we hypothesized that T_reg in 96tm-Tg mice represented a significant brake on the development of autoimmune diseases. If so, removal of such a brake might lead to exacerbation of systemic autoimmunity. We tested this hypothesis by injecting PC61 into 96tm-Tg mice beginning at the neonatal stage (within 9 days after birth) every 4 days for four injections. Down-regulation of CD25 was confirmed by flow cytometric analysis of peripheral blood 4 days after the last PC61 injection (data not shown). Inactivation of T_reg by this method was transient when PC61 was given neonatally. All mice fully recovered the number of T_reg in the peripheral blood as early as 1 wk after the last PC61 injection. PC61-treated 96tm-Tg mice were sacrificed at 5 mo for analysis of autoimmune disease, at which point the distribution of CD4⁺ T cells, CD8⁺ T cells, and B cells were comparable between mice treated with PC61 and mice treated with control Ig (data not shown). The percentage and cellularity of T_reg also returned normal (data not shown).

However, PC61-treated 96tm-Tg mice manifested a significant exacerbation of autoimmunity by the appearance of ANA at least 2 wk earlier (age 10 wk) than control Ig-treated mice (age 12 wk or older) (Fig. 5A) and the worsening of their immune complex-mediated glomerulonephritis (Fig. 5B). Furthermore, we found that the serum level of IL-12/IL-23 p40 subunit was significantly increased in PC61-treated mice (Fig. 5C), whereas serum IL-1, TNF-, and IL-6 remained undetectable in mice treated with PC61 or control Ig (data not shown). Given our recent finding that IL-12 was pathological in the lupus-like autoimmune diseases in 96tm-Tg mice (35), we conclude that T_reg inactivation in 96tm-Tg mice accelerates systemic autoimmune diseases.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Exacerbation of autoimmune diseases in 96tm-Tg mice after functional inactivation of Treg during their neonatal lives. Newborn mice (within 9 days after birth) were treated with PC61 or control IgG every 4 days for four injections. A, Mean fluorescence staining intensity (MFI) of ANA at week 10. B, Severity score of immune complex-mediated glomerulonephritis at week 20. C, Serum level of IL-12p40 at week 20. Symbols represent one individual mouse and horizontal bar represents the mean value of serum level.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Autoimmune gastritis in 96tm- Tg mice but not WT mice after Treg inactivation. Mice were treated as in Fig. 5. A, Gastric histology of WT and 96tm-Tg mice after treatment with PC61 or control Ab. One representative mouse is shown for each group. B, Quantification of severity of gastritis. Each symbol represents one individual mouse with horizontal bar representing average value of severity.

We have shown recently that 96tm is a chaperone for TLR4, resulting in hyperresponsiveness of 96tm-expressing cells to LPS (25). We further demonstrated that TLR4 is critical for the onset of autoimmunity in 96tm-Tg mice. Because direct TLR4 engagement on T_reg has been reported to enhance regulatory function of T_reg (17), we hypothesized that the enhanced T_reg activity in 96tm-Tg mice was the direct effect of increased chronic triggering of TLR4 due to 96tm expression on T_reg. This hypothesis necessitates the expression of 96tm by T_reg. As expected, we found that Tg T_reg but not WT T_reg expressed transcripts for 96tm (Fig. 7A). More importantly, we found that T_reg from TLR4 null 96tm-Tg mice lost their superior ability to inhibit the proliferation of T_eff (Fig. 7B), demonstrating that the increased suppression by 96tm-Tg T_reg in comparison to WT T_reg was mediated by TLR4. Our study is the first example of TLR4 hyperfunction in vivo in inducing increased T_reg function to curtail TLR4-dependent autoimmunity.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. TLR4 dependency for increased suppression by 96tm-Tg Treg. A, RT-PCR analysis for the expression of endogenous gp96, 96tm, and -actin mRNA from purified Treg and in vitro cultured bone marrow-derived macrophages (BMDM) of WT and 96tm-Tg origin B, In vitro suppression assay of Treg from WT, TLR4–/–, 96tm-TLR4+/–, or 96tm-TLR4–/– mice. WT Teff were stimulated with anti-CD3 Ab plus WT APC in the presence of various Treg, as shown in Fig. 3. Two independent experiments were performed with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Thymectomy on day 3 has been widely used to investigate the roles of T_reg in vivo. One enigma is that day 3 thymectomy leads to organ-specific rather than the systemic autoimmunity such as lupus. Depending upon mouse strain and gender, thymectomy on day 3 may induce inflammation in the stomach, thyroid, ovary, testicle, prostate, or other organs (38, 39). For example, C57BL/6 mice are resistant to the development of autoimmunity after thymectomy on day 3, including gastritis (38). Even in BALB/c mice that are more susceptible to day 3 thymectomy-induced gastritis (38, 40), inactivation of T_reg is necessary but not sufficient to induce autoimmune gastritis (31). Yet, inactivation of T_reg in our 96tm-Tg mice, which were in C57BL/6 background was sufficient to induce gastritis (Fig. 6), and exacerbate the systemic autoimmunity as indexed by the worsening of glomerulonephritis and earlier emergence of ANA (Fig. 5). Our finding demonstrated that T_reg exerts a significant brake in the development of autoimmune diseases in 96tm-Tg mice. It offers a striking example that T_reg can suppress both organ-specific and systemic autoimmunity in vivo.

The increased suppressive functions of T_reg from 96tm-Tg mice were directly demonstrated in vitro (Fig. 3). T_reg from Tg mice were able to exert much more suppression on the proliferation of either WT or Tg T_eff regardless whether 96tm was expressed on APCs. Moreover, unlike WT mice that have age-dependent reduction of T_reg activity (8, 26, 27), we found no decline of Tg T_reg function with age (Fig. 3C). The increased Tg T_reg function was also indirectly demonstrated in vivo, as we observed much more robust and systemic expansion and survival of SEA-specific Tg CD4⁺ T cells after the inactivation of T_reg (Fig. 4).

T_reg need to be activated before they can function. This requires IL-2 (5, 41) and costimulation, such as B7-CD28/CTLA-4 interaction (6, 42). Additional signals capable of regulating the T_reg activity remain elusive. TLR are ancient molecules that detect microbial products and differentiate "non-self" from "self" (43). Engagement of TLRs with their ligands induces the activation of innate immunity. As a consequence, the TLR-induced proinflammatory environment can further promote the activation of the adaptive immunity (44). Recently, the roles of TLRs in regulating the functions of T_reg have gained increasing attention (18). In response to LPS, mouse DCs produces IL-6, which is partially responsible for releasing T_eff from T_reg inhibition (16). In contrast, reversal of T_reg function could be DC-independent (13). For example, TLR8 activation dampens the functions of T_reg directly, which is dependent on MyD88-IRAK4 signaling pathway in the T_reg autonomous fashion. Moreover, it has been shown that T_reg selectively express TLR4 (17) and that interaction of T_reg with LPS induces their suppressive function. In another study, TLR2 signaling was shown to promote the survival of CD4⁺CD25⁺ T_reg as these cells were significantly decreased in TLR2^–/– mice (45). More recently, heat shock protein 60 was reported to be able to enhance the function of human T_reg in vitro via TLR2 in a manner that is dependent on both cell-cell contact and immunosuppressive cytokines IL-10 and TGF- (46). Our present work is consistent with the roles of TLR4 activation in directly enhancing T_reg function (17, 18), providing a strong piece of in vivo evidence that excessive TLR activation could be counter-balanced by amplified T_reg function.

Several unanswered questions remain. For example, what are the differences if any in the mechanisms of suppression between WT and Tg T_reg? Our analyses so far have found no discernible distinctions between the two, including their ability to proliferate in vitro, the activation status measured by the level of CD3 chain phosphorylation, the cell surface marker of CD103 (47) (data not shown), and their cytokine profile in response to polyclonal activation (Fig. 2). Secondly, the number of T_reg in WT and Tg is similar. The coordinated actions of Foxp3 and NFAT are critical for the development of T_reg (4), yet it is unclear whether expression of gp96 on cell surface might affect this process qualitatively. Thirdly, there are many flavors of T_reg in vivo (48). In particular, natural Foxp3⁺ T_reg can be converted into inducible T_reg in the presence of appropriate environment such as the presence of TGF- and IL-10. It is unclear whether these processes were in someway altered in 96tm-Tg mice.

In summary, we have shown that cell surface expression of a TLR-chaperone, gp96, induced lupus-like diseases and elevated T_reg function without altering the development of CD4⁺CD25⁺ Foxp3⁺ T_reg. Our study represents the first example of curtailing TLR4 hyperactivation via T_reg on the organismal level to prevent systemic autoimmune diseases. Further studies into the mechanisms of this process in the context of infection, tissue injury, inflammation, and autoimmune pathology should be critical and fruitful in guiding the development of new immunotherapeutics against these conditions.

	Acknowledgments

 
We thank Drs. Leo Lefrançois, Mark Mamula, and Robert Clark for helpful discussions; Yi Yang and Matthew Staron (both from Dr. Zihai Li’s laboratory) for stimulating interactions during the course of the study as well as critical analysis of the data; and Dr. Mark Goldstein for reading and editing of the manuscript.

	Disclosures

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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 in part by the National Institutes of Health Grants CA100191 (to Z.L.) and AI 42858 (to A.T.V.). Z.L. is a clinical scholar of the Leukemia and Lymphoma Society.

² Address correspondence and reprint requests to Dr. Zihai Li, Center for Immunotherapy of Cancer and Infectious Diseases, Department of Immunology, University of Connecticut School of Medicine, Mail Code 1601, 263 Farmington Avenue, Farmington, CT 06030-1601. E-mail address: zli{at}up.uchc.edu

³ Abbreviations used in this paper: T_reg, regulatory T cell; T_eff, effector T cell; DC, dendritic cell; ANA, antinuclear Ab; GITR, glucocorticoid-induced TNFR; SEA, staphylococcal enterotoxin A; WT, wild type; Tg, transgenic.

Received for publication October 2, 2006. Accepted for publication December 8, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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