INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Allergic inflammation is characterized by a prominent eosinophil infiltrate (1). In addition to the infiltration of eosinophils, there is an increase in CD4⁺ T cells and interleukin-5 (IL-5)- producing cells at the sites of allergic late-phase reaction and allergic airway inflammation in asthma (2), suggesting that CD4⁺ T cells and their cytokine IL-5 might be involved in antigen-induced eosinophil recruitment into the tissue. Furthermore, in a murine model of airway late-phase reaction we and others have provided direct evidence that CD4⁺ T cells and IL-5 mediate antigen-induced eosinophil recruitment into the tissue of sensitized mice (5, 6). Thus, the manipulation for inducing selective inactivation of antigen-specific Th2 cells is a rational approach to the control of allergic inflammation.

It has recently been shown that CD4⁺ CD25⁺ T cells are immunoregulatory T cells that prevent induction of organ-specific autoimmune diseases (7). They constitutively express CD25 (IL-2 receptor -chain) and constitute 5 to 10% of peripheral CD4⁺ T cells in nonimmunized naive mice (7). In vitro, CD4⁺ CD25⁺ T cells inhibit the proliferation of CD4⁺ CD25 T cells (12, 13). The inhibition is not mediated by cytokines such as IL-4, IL-10, and transforming growth factor (TGF)- (12, 13) but is dependent on contact between responding CD4⁺ CD25 T cells and the CD4⁺ CD25⁺ T cells (12, 13). Among cell surface molecules, it has recently been shown that the expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on CD4⁺ CD25⁺ T cells is required for their regulatory function (14, 15).

In vivo, elimination of CD4⁺ CD25⁺ T cells results in the development of various organ-specific autoimmune diseases (7). CD4⁺ CD25⁺ T cells also suppress the autoimmunity in neonatally thymectomized mice (8, 9). Furthermore, CD4⁺ CD25⁺ T cells prevent the onset of autoimmune diabetes in nonobese diabetic (NOD) mice (16). However, the regulatory role of CD4⁺ CD25⁺ T cells in Th2 cell-mediated allergic inflammation is still unknown.

Therefore, to determine whether CD4⁺ CD25⁺ T cells downregulate Th2 cell-mediated allergic inflammation in the airways, we studied antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2^/ mice transferred with CD4⁺ CD25⁺ T cell-depleted or unfractionated T cells from nonimmunized ovalbumin-specific TCR transgenic mice. We also studied the effect of depletion of CD4⁺ CD25⁺ T cells on Th2 cell differentiation in vitro. Our results indicate that CD4⁺ CD25⁺ T cells upregulate Th2 cell-mediated antigen-induced eosinophil recruitment into the airways by modulating the T helper cell differentiation toward Th2 type.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mice

BALB/c mice and BALB/c SCID mice were purchased from Japan SLC (Shizuoka, Japan). Ovalbumin (OVA)-specific DO11.10 (DO10) TCR transgenic mice (17) were backcrossed to BALB/c mice for more than 10 generations. Rag-2^/ mice, which lack mature T cells and B cells (18), were also backcrossed for more than 10 generations onto BALB/c mice (BALB/c Rag-2^/ mice). Mice were housed in microisolator cages under pathogen-free conditions. All experiments were performed according to the guidelines of the National Institutes of Health, Bethesda, MD.

Depletion of CD4⁺ CD25⁺ T Cells from Splenocytes of DO10 Transgenic Mice

Single cell suspension of splenocytes was prepared from 8- to 10-wk-old nonimmunized DO10 transgenic mice. Cells were incubated with biotinylated anti-CD25 antibody (clone PC61; Immunotech, Marseilles, France) at 4° C for 20 min in the presence of anti-CD16/32 antibody (2.4G2; PharMingen, San Diego, CA) to block Fc receptors. After washing with phosphate-buffered saline (PBS), cells were incubated with streptavidin-magnetic microbeads (Miltenyi Biotec, Sunnyvale, CA) at 4° C for 20 min, washed three times with PBS, and passed through the MACS separation BS column (Miltenyi Biotec) according to the manufacturer's instructions. As a control, cells were incubated with isotype-matched biotinylated antibody (PharMingen) in the presence of anti-CD16/32 antibody, followed by incubation with streptavidin-magnetic microbeads, and passed through the BS column. The efficiency of depletion was evaluated by FACS with anti-CD4 APC and anti-CD25 FITC (clone 7D4; PharMingen), which differentiates the epitopes of murine CD25 from that of PC61 (19). Over 99% of CD4⁺ CD25⁺ T cells was removed from splenocytes by this procedure (see Figure 2).

View larger version (25K): [in this window] [in a new window]   Figure 2.   Depletion of CD4+ CD25+ T cells from splenocytes of nonimmunized DO10 mice. CD4+ CD25+ T cells were depleted from splenocytes of nonimmunized DO10 mice by magnetic cell sorting using anti-CD25 antibody (clone PC61) (right panel ) as described in METHODS. The efficiency of depletion was evaluated by FACS with anti-CD4 APC and anti-CD25 FITC (clone 7D4), which recognizes the different epitope of murine CD25 from that of PC61. Over 99% of CD4+ CD25+ T cells was removed from splenocytes by this procedure.

Purification of CD4⁺ CD25⁺ T Cells from Nonimmunized DO10 Mice

Single cell suspension of splenocytes was prepared from 8- to 10-wk-old DO10 transgenic mice. Cells were incubated with a mixture of FITC-labeled antibodies to B220 (R43-6B2, PharMingen), pan NK (DX5; PharMingen), CD8 (53.6.7; PharMingen), and CD11b (Mac-1, M1/70; PharMingen) for 20 min at 4° C, washed three times with PBS, and incubated with anti-FITC magnetic microbeads (Miltenyi Biotec) for 20 min at 4° C. After washing with PBS, cells were passed through the MACS separation CS column (Miltenyi Biotec) and cells in the flow-through were collected by centrifugation. At this point, these cells were > 90% pure CD4⁺ T cells by FACS analysis. Subsequently, collected cells were incubated with anti-CD25 (7D4) biotin for 20 min at 4° C, washed three times with PBS, and incubated with streptavidin-magnetic microbeads for 20 min at 4° C. After washing with PBS, CD25⁺ cells were positively collected by MACS RS⁺ column (Miltenyi Biotec) (CD4⁺ CD25⁺ T cells), and CD25 cells were negatively collected by passing through a MACS AS column (Miltenyi Biotec) (CD4⁺ CD25 T cells). The purity of CD4⁺ CD25⁺ T cells and of CD4⁺ CD25 T cells was > 85% and > 95%, respectively.

Cell Transfer System for Antigen-induced Eosinophil and T Cell Infiltration in Mouse Airways

Either CD25⁺ T cell-depleted splenocytes (1 × 10⁷ cells/mouse), unfractionated splenocytes (1 × 10⁷ cells/mouse), or a mixture of CD25⁺ T cell-depleted splenocytes (1 × 10⁷ cells/mouse) and purified CD4⁺ CD25⁺ T cells (1.5 × 10⁵ cells/mouse) from DO10 mice were injected intravenously to BALB/c Rag-2^/ mice at Day 0. These mice were then immunized twice with 4 µg of OVA (Sigma, St. Louis, MO) in 4 mg of aluminum hydroxide intraperitoneally at Day 1 and Day 15. Fourteen days after the second immunization, the sensitized mice were given aerosolized OVA (50 mg/ml) dissolved in 0.9% saline by a DeVilbiss 646 nebulizer (DeVilbiss Corp., Somerset, PA) for 20 min. As a control, 0.9% saline alone was administered by the nebulizer. At 24 or 36 h after the inhalation, the trachea was excised, fixed in 10% buffered formalin, and embedded in paraffin and the specimens (3 µm thick) were stained with Luna solution and hematoxylin-eosin solution. The number of eosinophils in the submucosal tissue of trachea was counted in Luna-stained sections and expressed as the number of eosinophils per the length of the basement membrane of trachea, which was measured with a digital curvimeter (5). In preliminary experiments, we found that the immunization with OVA was required for the induction of antigen-induced eosinophil recruitment into the airways in this system.

The eosinophil and T cell infiltration into the bronchoalveolar lavage fluid (BALF) was also evaluated as described previously (20). In brief, bronchoalveolar lavage was performed with 1.2 ml of PBS at 36 h after saline or OVA inhalation. After BALF cells were counted using a hemocytometer, differential cell counts were performed on cytospin cell preparations stained with Wright-Giemsa solution. A fraction of the cells was subjected to a flow cytometric analysis for the lymphocyte surface phenotyping as described below.

Cytokine Levels in BALF

The BALF was centrifuged at 400 × g for 5 min at 4° C, and the amounts of IL-2, IL-4, IL-5, and interferon (IFN)- in the supernatant were determined by enzyme immunoassay using murine IL-2, IL-4, IL-5, and IFN- ELISA kits (PharMingen). The assays were performed in duplicate according to the manufacturer's instruction. The minimum significant values of these assays were 10 pg/ml of IL-2, IL-4, and IL-5, and 50 pg/ml of IFN-.

Antigen-Induced Cytokine Production in Splenocyte Culture

After CD4⁺ CD25⁺ T cells were removed from DO10 splenocytes as described above, cells (2 × 10⁵) were suspended in 200 µl of RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 µM 2-mercaptoethanol, 2 mM L-glutamine, and antibiotics and were cultured in triplicate in the absence or presence of OVA323-339 peptide (0.5 µg/ml) in a 96-well microtiter plate at 37° C for 48 h. As a control, unfractionated splenocytes (2 × 10⁵) were cultured in the same condition. The culture supernatant was collected and the amount of IL-2, IL-4, IL-5, and IFN- was measured by enzyme immunoassay as described above.

Flow Cytometric Analysis

Cells from the BALF and spleen were stained and analyzed on a FACScalibur (Becton Dickinson, San Jose, CA) using CELLQuest software (21). For direct staining, the following conjugated antibodies were purchased from PharMingen: anti-CD4 FITC, PE, PerCP, APC (H129.19), anti-B220 FITC (RA3-6B2), anti-CD8 FITC (53.6.7), anti-CD11b (Mac-1) FITC (M1/70), anti-CD25 PE (PC61), anti-CD25 FITC (7D4), anti-CD69 PE (H1.2F3), anti-Pan NK FITC (DX5), anti-IL-2R FITC (TM-1), and anti-_c PE (4G2). KJ1-26 monoclonal antibody (mAb), antiidiotype for DO10 TCR (22) was purified from supernatants of hybridoma cells using protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC or biotin. Prior to staining, Fc receptors were blocked with anti-CD16/32 antibody (2.4G2).

Intracellular Staining for IL-4 and IFN-

CD4⁺ CD25⁺ T cell-depleted or unfractionated DO10 splenocytes (1 × 10⁶) were stimulated with OVA323-339 peptide (0.5 µg/ml) in a 24-well microtiter plate at 37° C for 48 h. Where indicated, IL-12 (7.5 ng/ml; R&D Systems Inc., Minneapolis, MN) was added to polarize toward Th1 cells (Th1 condition) and IL-4 (7.5 ng/ml; R&D Systems Inc.) was added to polarize toward Th2 cells (Th2 condition). Cells were washed with PBS and cultured for another 3 d in either Th0 (nonpolarizing), Th1, or Th2 condition in the presence of IL-2 (15 ng/ml; R&D Systems Inc.). Cells were then restimulated with plate-bound anti-CD3 antibody for 6 h, with monensin (2 µM) (Sigma) added for the final 4 h to prevent cytokine release. Cells were harvested, washed with PBS, and stained with anti-CD4 PerCP for 30 min at 4° C. Cells were washed with PBS, fixed with IC FIX (BioSource International, Inc., Camarillo, CA), permeabilized with IC PERM (BioSource International, Inc.), and stained with anti-IL-4 PE (BVD4-1D11; PharMingen) and anti-IFN- APC (XMG1.2; PharMingen) for 30 min at 4° C. After washing, cells were analyzed on a FACScalibur using CELLQuest software. Negative controls consisted of isotype-matched, directly conjugated, nonspecific antibodies (PharMingen).

Determination of Antigen-specific IgE Antibody in Serum

At 2 wk after the second immunization, the titer of OVA-specific immunoglobulin E (IgE) antibody in mouse serum was assessed by a 24-h passive cutaneous anaphylaxis (PCA) reaction as described previously (21).

Data Analysis

Data are summarized as mean ± SD. The statistical analysis of the results was performed by the unpaired t test. p Values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CD4⁺ CD25⁺ T Cells in Nonimmunized DO10 Mice Differ from Naturally Activated CD4⁺ T Cells

It has recently been shown that CD4⁺ CD25⁺ T cells are immunoregulatory T cells that prevent CD4⁺ T cell-mediated organ-specific autoimmune diseases (7). We first evaluated the characteristic of CD4⁺ CD25⁺ T cells in nonimmunized DO10 mice. Consistent with a previous finding (13), approximately 6% of CD4⁺ T cells in spleen expressed CD25 in nonimmunized mice (Figure 1A). The majority of CD4⁺ CD25⁺ T cells and CD4⁺ CD25 T cells expressed transgenic TCR (KJ1-26⁺) (data not shown). Regarding other IL-2 receptor components, CD4⁺ CD25⁺ T cells expressed the common cytokine receptor  chain (_c), but lacked IL-2R chain (data not shown), suggesting that these cells do not express functional IL-2 receptors, which are composed of a heterodimer of IL-2R and _c or a heterotrimer of CD25, IL-2R, and _c (23). As shown in Figure 1B, the size of CD4⁺ CD25⁺ T cells in nonimmunized mice, which was evaluated by forward light scatter of FACS analysis, was similar to that of CD4⁺ CD25 T cells. When purified CD4⁺ CD25⁺ T cells from nonimmunized mice were stimulated with anti-CD3 antibody for 6 h, no significant number of IL-4-producing cells and a small number of IFN--producing cells were detected in the culture (Figure 1D) and the cytokine profiles (IL-4 versus IFN-) were indistinguishable between CD4⁺ CD25⁺ T cells and CD4⁺ CD25 T cells (Figure 1D versus 1C). Moreover, few IL-5-producing cells were detected in the culture of anti-CD3-stimulated CD4⁺ CD25⁺ T cells by intracellular cytokine analysis (data not shown). These observations suggest that the majority of CD4⁺ CD25⁺ T cells in nonimmunized DO10 mice differ from "naturally activated" CD4⁺ T cells (24), because it has been shown that "naturally activated" CD4⁺ T cells are large in size and rapidly produce a large amount of cytokines upon activation (24).

View larger version (40K): [in this window] [in a new window]   Figure 1.   CD4+ CD25+ T cells in nonimmunized DO10 transgenic mice do not produce IL-4 on activation. (A) CD4 versus CD25 staining of CD4+ T cells in DO10 splenocytes. Splenocytes from OVA-specific DO10 TCR transgenic mice were stained with anti-CD4 FITC and anti-CD25 PE and analyzed on FACScalibur. Shown is a representative CD4 versus CD25 staining on CD4+ T cells in nonimmunized DO10 mice (n = 10). (B) Cell size of CD4+ CD25+ T cells. Splenocytes from nonimmunized DO10 mice were stained with anti-CD4 FITC and anti-CD25 PE and cell size of CD4+ CD25+ T cells (thick line histogram) and CD4+ CD25 T cells (dot line histogram) was evaluated by forward light scatter (FSC). Data are shown on a linear scale. (C, D) Cytokine production of CD4+ CD25+ T cells. CD4+ CD25+ T cells and CD4+ CD25 T cells were purified as described in METHODS. Cells were stimulated with plate-bounded anti-CD3 antibody for 6 h, with monensin (2 mM) added for the final 4 h. Intracellular cytokine profiles (IL-4 versus IFN-) on CD4+ T cells were evaluated as described in METHODS.

Depletion of CD4⁺ CD25⁺ T Cells Decreases Antigen-induced Eosinophil Recruitment into the Airways

To determine whether CD4⁺ CD25⁺ T cells downregulate Th2-type CD4⁺ T cell-mediated allergic inflammation in the airways (5), we evaluated antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2^/ mice transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes or unfractionated splenocytes from nonimmunized DO10 mice. We used the adoptive cell transfer system of BALB/c Rag-2^/ mice, which lack T cells and B cells, as recipients to eliminate the influence of immune responses of host cells, because CD25 is expressed not only on CD4⁺ CD25⁺ regulatory T cells but also on conventional T cells upon activation.

As shown in Figure 2, over 99% of CD4⁺ CD25⁺ T cells were eliminated from splenocytes of nonimmunized DO10 mice by magnetic cell sorting using anti-CD25 antibody. In contrast, other cell types including CD4⁺ CD25 T cells, CD8⁺ cells, B220⁺ cells, Mac-1⁺ cells, and DX5⁺ cells (NK cells) were not eliminated (data not shown). After BALB/c Rag-2^/ mice were transferred intravenously with CD4⁺ CD25⁺ T cell-depleted splenocytes or unfractionated splenocytes (as a control) from DO10 mice, these mice were immunized intraperitoneally twice with OVA at a 2-wk interval and challenged with inhaled OVA or saline (as a negative control) at 2 wk after the second immunization.

The inhalation of aerosolized OVA caused the infiltration of eosinophils into the trachea of BALB/c Rag-2^/ mice when unfractionated splenocytes from DO10 mice were pretransferred to the mice (Figure 3). This antigen-induced eosinophil recruitment in the trachea was not observed when CD4⁺ T cell-depleted splenocytes from DO10 mice were transferred to BALB/c Rag-2^/ mice (data not shown), indicating that the antigen-induced eosinophil recruitment into the trachea was mediated by CD4⁺ T cells from DO10 mice in this system. In addition, in vivo administration of recombinant IFN- before the inhaled antigen challenge decreased the antigen-induced eosinophil recruitment into the airways (data not shown), which is consistent with our previous findings that the administration of IFN- inhibits the airway eosinophilia in OVA-sensitized wild-type mice (25). These results suggest that the mechanism underlying eosinophil recruitment into the airways in this system is similar to that in OVA-sensitized wild-type mice.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Depletion of CD4+ CD25+ T cells decreases antigen-induced eosinophil recruitment into the mouse airways. (A) BALB/c Rag-2/ mice were transferred intravenously with CD4+ CD25+ T cell-depleted splenocytes or unfractionated splenocytes from nonimmunized DO10 mice. The mice were then immunized intraperitoneally twice with OVA and 2 wk after the second immunization, the mice were challenged with inhaled OVA or saline (as a negative control). At 24 or 36 h after the inhalation, antigen-induced eosinophil recruitment into the trachea was evaluated as described in METHODS. Data are means ± SD for seven mice in each group. *Significantly different from the mean value of the corresponding response in the mice that were transferred with unfractionated splenocytes, *p < 0.001. (B) Similar to A. BALB/c Rag-2/ mice were transferred with CD4+ CD25+ T cell-depleted splenocytes together with (hatched columns) or without (open column) purified CD4+ CD25+ T cells (at 5% of CD4+ CD25 T cells). As controls, BALB/c Rag-2/ mice were transferred with unfractionated splenocytes (closed columns). These mice were then immunized and challenged with OVA, and antigen-induced eosinophil recruitment into the trachea was evaluated. Data are means ± SD for five mice in each group. *Significantly different from the mean value of the corresponding response without adding back purified CD4+ CD25+ T cells, *p < 0.005.

However, contrary to our expectations, antigen-induced eosinophil recruitment into the trachea was significantly decreased when BALB/c Rag-2^/ mice were transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes (unfractionated 3.3 ± 0.8 versus CD25-depleted 0.8 ± 0.4 eosinophils/mm at 24 h after OVA inhalation, mean ± SD, n = 7 mice in each group, p < 0.001) (Figure 3A). Similar results were also obtained when BALB/c SCID mice were used as recipient mice (data not shown), suggesting that the finding is not restricted to the Rag-2^/ mice. In addition, cotransfer of purified CD4⁺ CD25⁺ T cells at the physiological ratio (5% of CD4⁺ CD25 T cells) to BALB/c Rag-2^/ mice with CD4⁺ CD25⁺ T cell-depleted splenocytes significantly restored decreased antigen-induced eosinophil recruitment into the airways (n = 5 mice, p < 0.005) (Figure 3B). Moreover, the number of eosinophils recovered from BALF after antigen inhalation was significantly decreased when BALB/c Rag-2^/ mice were transferred with CD25⁺ T cell-depleted splenocytes (unfractionated 3.5 ± 0.8 versus CD25-depleted 1.6 ± 0.4 x10⁴ at 36 h after OVA inhalation, n = 5 mice in each group, p < 0.001) (Figure 4A). These results indicate that CD4⁺ CD25⁺ T cells enhance Th2 cell-mediated antigen-induced eosinophil recruitment into the airways.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Depletion of CD4+ CD25+ T cells increases antigen-induced neutrophil and T cell recruitment into the airways. BALB/c Rag-2/ mice were transferred with CD4+ CD25+ T cell-depleted or unfractionated splenocytes from nonimmunized DO10 mice, immunized twice with OVA, and then challenged with inhaled OVA or saline. The number of eosinophils (a), neutrophils (b), and lymphocytes (c) recovered in the BALF was then counted at 36 h after the inhalation. Data are means ± SD for five mice in each group. *Significantly different from the mean value of the corresponding response in the mice that were transferred with unfractionated splenocytes, *p < 0.001.

Depletion of CD4⁺ CD25⁺ T Cells Increases Antigen-induced Neutrophil and T Cell Recruitment into the Airways

On the other hand, antigen-induced neutrophil and T cell recruitment in the airways was significantly increased when BALB/c Rag-2^/ mice were transferred with CD25⁺ T cell-depleted splenocytes. The number of neutrophils in BALF after antigen inhalation, which has been reported to be a characteristic of Th1 cell-mediated inflammation (26), was significantly increased by the depletion of CD4⁺ CD25⁺ T cells (unfractionated 37.6 ± 9.8 versus CD25-depleted 75.7 ± 12.7 × 10⁴, n = 5, p < 0.001) (Figure 4B). The number of lymphocytes in BALF was also significantly increased by the depletion of CD4⁺ CD25⁺ T cells (n = 5, p < 0.001) (Figure 4C).

FACS analysis of BALF cells revealed that the recruitment of OVA-specific CD4⁺ KJ1-26⁺ T cells in the airways was significantly increased by antigen inhalation in mice that were transferred with CD25⁺ T cell-depleted splenocytes (unfractionated 13.9 ± 3.2 versus CD25-depleted 42.8 ± 12.4 x10⁴, n = 5 mice, p < 0.001) (Figure 5A). However, the expression of CD69 on CD4⁺ KJ1-26⁺ T cells was similarly upregulated after antigen inhalation (Figure 5B), indicating that CD4⁺ KJ1-26⁺ T cells were equally activated by antigen inhalation regardless of the presence or absence of CD4⁺ CD25⁺ T cells. Collectively, these results, that depletion of CD4⁺ CD25⁺ T cells deceased Th2 cell-mediated eosinophil recruitment (5, 26) but increased Th1 cell-mediated neutrophil recruitment, suggest that the depletion of CD4⁺ CD25⁺ T cells induces Th1-biased immune responses in vivo.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Depletion of CD4+ CD25+ T cells increases antigen-specific T cell recruitment into the airways. BALB/c Rag-2/ mice were transferred with CD4+ CD25+ T cell-depleted or unfractionated splenocytes from nonimmunized DO10 mice, immunized twice with OVA, and then challenged with inhaled OVA or saline. The number of KJ1-26+ CD4+ T cells in BALF (A) was then counted at 36 h after the inhalation. At the same time, the expression of CD69 (B) on KJ1-26+ CD4+ T cells was analyzed by FACS. Data are means ± SD for five mice in each group. *Significantly different from the mean value of the corresponding response in the mice that were transferred with unfractionated splenocytes, *p < 0.001.

Depletion of CD4⁺ CD25⁺ T Cells Decreases Antigen-induced IL-4 and IL-5 Production in the Airways

To determine whether CD4⁺ CD25⁺ T cells induce the immune deviation of Th1 and Th2 cytokines in vivo, we examined antigen-induced cytokine production in the airways of these mice. Consistent with the decreased antigen-induced eosinophil recruitment into the airways (Figures 3 and 4), antigen-induced IL-4 and IL-5 production in the airways was significantly decreased when BALB/c Rag-2^/ mice were transferred with CD25⁺ T cell-depleted splenocytes (IL-4: unfractionated 56.4 ± 14.5 versus CD25-depleted < 10 pg/ml, n = 6, p < 0.001; IL-5: unfractionated 107.2 ± 23.5 versus CD25-depleted 50.6 ± 12.5 pg/ml, n = 5, p < 0.01) (Table 1). In contrast, antigen-induced IFN- production in the airways was not significantly affected by the depletion of CD4⁺ CD25⁺ T cells (Table 1). No significant amount of antigen-induced IL-2 production was detected in BALF of these mice. These results indicate that the depletion of CD4⁺ CD25⁺ T cells decreases antigen-induced Th2 cytokine production in the airways and thus decreases antigen-induced eosinophil recruitment into the airways.

Depletion of CD4⁺ CD25⁺ T Cells Decreases Antigen-specific IgE Production

To determine whether CD4⁺ CD25⁺ T cells are involved in the regulation of antigen-specific IgE production, another parameter of Th2-type immune response, we measured the titer of OVA-specific IgE in these mice. Upon immunization with OVA, OVA-specific IgE was readily detectable in BALB/c Rag-2^/ mice that were transferred with unfractionated splenocytes from DO10 mice (Figure 6). In contrast, OVA-specific IgE was significantly decreased in BALB/c Rag-2^/ mice that were transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes (n = 7 mice, p < 0.001) (Figure 6). These results indicate that CD4⁺ CD25⁺ T cells play a significant role in the production of antigen-specific IgE antibody in this system.

View larger version (12K): [in this window] [in a new window]   Figure 6.   Depletion of CD4+ CD25+ T cells decreases antigen-specific IgE production. BALB/c Rag-2/ mice were transferred intravenously with CD4+ CD25+ T cell-depleted splenocytes or unfractionated splenocytes from nonimmunized DO10 mice. The mice were then immunized intraperitoneally twice with or without OVA and the sera were collected for OVA-specific IgE determination at 2 wk after the second immunization. OVA-specific IgE was evaluated by passive cutaneous anaphylaxis as described in METHODS. Data are means ± SD for seven mice in each group. *Significantly different from the mean value of the corresponding response in the mice that were transferred with unfractionated splenocytes, *p < 0.001.

Depletion of CD4⁺ CD25⁺ T Cells Prevents Th2 Cell Differentiation In Vitro

To determine whether CD4⁺ CD25⁺ T cells control the differentiation of Th1 and Th2 cells, we examined the effect of CD4⁺ CD25⁺ T cell depletion on antigen-induced cytokine production from CD4⁺ T cells in vitro. Interestingly, the depletion of CD4⁺ CD25⁺ T cells significantly increased antigenic peptide (OVA323- 339)-induced IL-2 production (unfractionated 2608.0 ± 523.0 versus CD25-depleted 5347.0 ± 622.7 pg/ml, n = 6 mice in each group, p < 0.001) as well as IFN- production (unfractionated 12.21 ± 2.30 versus CD25-depleted 22.33 ± 3.06 ng/ml, n = 6, p < 0.001) from DO10 splenocytes (Table 2). In contrast, OVA323-339 peptide-induced IL-4 and IL-5 production from DO10 splenocytes was significantly decreased by the depletion of CD4⁺ CD25⁺ T cells (IL-4: unfractionated 214.2 ± 57.8 versus CD25-depleted 27.2 ± 6.5 pg/ml, p < 0.001; IL-5: unfractionated 68.3 ± 9.3 versus CD25-depleted 38.7 ± 14.3 pg/ml, p < 0.01) (Table 2).

We further examined the effect of CD4⁺ CD25⁺ T cell depletion on antigen-induced cytokine production from CD4⁺ T cells at single cell levels. Consistent with the results in Table 2, the depletion of CD4⁺ CD25⁺ T cells significantly decreased the differentiation of Th2 (IL-4⁺ IFN-) cells (unfractionated 31.5% versus CD25 depleted 2.7%) (Figure 7A). In contrast, Th1 (IL-4 IFN-⁺) cells were significantly increased by the depletion of CD4⁺ CD25⁺ T cells (unfractionated 13.7% versus CD25 depleted 28.0%) (Figure 7A). As expected, when purified CD4⁺ CD25⁺ T cells were cocultured with CD4⁺ CD25⁺ T cell-depleted splenocytes at the physiological ratio, the balance between Th1 cells and Th2 cells was restored to that of unfractionated splenocytes (Figure 7A). These results indicate that CD4⁺ CD25⁺ T cells modulate the T helper cell differentiation toward Th2 cells.

View larger version (50K): [in this window] [in a new window]   Figure 7.   Depletion of CD4+ CD25+ T cells prevents Th2 cell differentiation in vitro. (A) CD4+ CD25+ T cell-depleted splenocytes from DO10 mice were stimulated with OVA323-339 peptide (0.5 µg/ml) for 48 h in the presence or absence of purified CD4+ CD25+ T cells (at 5% of CD4+ CD25 T cells). As a control, unfractionated splenocytes from DO10 mice were stimulated with OVA323-339 peptide for 48 h. Cells were cultured in the presence of IL-2 for another 3 d and then restimulated with plate-bound anti-CD3 antibody for 6 h. Intracellular staining for IL-4 and IFN- on CD4+ T cells was then analyzed by FACS as described in METHODS. Shown are representative FACS profiles from five mice in each group. (B) CD4+ CD25+ T cell-depleted or unfractionated splenocytes were stimulated with OVA323-339 peptide (0.5 µg/ ml) for 48 h in the Th1- or Th2-polarizing condition. Cells were cultured in the presence of IL-2 for another 3 d and then restimulated with plate-bound anti-CD3 antibody for 6 h. Intracellular staining for IL-4 and IFN- on CD4+ T cells was then analyzed as described above. Shown are representative FACS profiles from five mice in each group.

Finally, we determined the effect of Th1 or Th2 polarization on the T helper cell differentiation in the presence or absence of CD4⁺ CD25⁺ T cells. As shown in the upper panels of Figure 7B, Th1 cell differentiation still dominated in the absence of CD4⁺ CD25⁺ T cells in the Th1-polarizing condition (unfractionated 42.6% versus CD25-depleted 71.7%). In contrast to Th1 cell differentiation, however, no significant difference was observed in the differentiation of Th2 cells between unfractionated and CD25⁺ T cell-depleted splenocytes in the Th2-polarizing condition (Figure 7B). Taken together with in vivo experiments, these results suggest that the impairment of Th2 cell differentiation by the depletion of CD4⁺ CD25⁺ T cells may be due to the inhibitory effect of CD4⁺ CD25⁺ T cells on Th1 cell differentiation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we show that CD4⁺ CD25⁺ T cells upregulate Th2 cell-mediated allergic inflammation in the airways and that CD4⁺ CD25⁺ T cells enhance the differentiation of Th2 cells. We found that Th2 cell-mediated antigen-induced eosinophil recruitment into the airways was significantly decreased in BALB/c Rag-2^/ mice that were transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes as compared with those transferred with unfractionated splenocytes (Figures 3 and 4). In addition, the depletion of CD4⁺ CD25⁺ T cells decreased antigen-specific IgE production (Figure 6). On the other hand, the depletion of CD4⁺ CD25⁺ T cells increased antigen-induced neutrophil recruitment in the mouse airways (Figure 4), which is characteristic of Th1 cell-mediated inflammation (26). We also found that the depletion of CD4⁺ CD25⁺ T cells decreased antigen-induced IL-4 and IL-5 production in the airways of the mice (Table 1). Finally, we found that the depletion of CD4⁺ CD25⁺ T cells prevented antigen-induced Th2 cell differentiation but increased Th1 cell differentiation in vitro (Figure 7). These results indicate that CD4⁺ CD25⁺ T cells modulate the T helper cell differentiation toward Th2 cells and thus upregulate Th2 cell-mediated allergic inflammation in the airways.

Our results indicate that CD4⁺ CD25⁺ T cells are essential for the appropriate Th2 cell differentiation. Because the differentiation of Th1 cells and Th2 cells is reciprocally controlled by their cytokines, the increased Th2 cell differentiation in the presence of CD4⁺ CD25⁺ T cells could result from either the enhancement of Th2 cell differentiation or the inhibition of Th1 cell differentiation by CD4⁺ CD25⁺ T cells. However, we believe the latter for the following reasons. First, previous studies showed that IL-2, IL-4, IL-10, or TGF- was not detectable in the culture supernatants of anti-CD3-stimulated CD4⁺ CD25⁺ T cells and that these cytokines were not essential for the regulatory function of CD4⁺ CD25⁺ T cells (12, 13). We also found that when CD4⁺ CD25⁺ T cells in nonimmunized DO10 mice were stimulated with anti-CD3 antibody, few IL-4-producing cells were detected by intracellular cytokine staining (Figure 1). These findings suggest that the Th2 cytokine profile induced by the presence of CD4⁺ CD25⁺ T cells was not due to the cytokine production by CD4⁺ CD25⁺ T cells themselves and that cytokines produced by CD4⁺ CD25⁺ T cells may not significantly affect the differentiation of CD4⁺ CD25 T cells to Th2 cells. Second, it has been demonstrated that CD4⁺ CD25⁺ T cells are nonproliferative but inhibit the proliferation of CD4⁺ CD25 T cells in a cell contact-dependent fashion (12, 13). Third, we found that antigen-induced T cell recruitment into the airways was increased by the depletion of CD4⁺ CD25⁺ T cells (Figure 5). Collectively, these findings suggest that CD4⁺ CD25⁺ T cells enhance Th2 cell differentiation by preferentially inhibiting the activation of Th1-type CD4⁺ CD25 T cells.

We also show that CD4⁺ CD25⁺ T cells play a significant role in antigen-specific IgE production in this system. We found that OVA-specific IgE was significantly decreased when Rag-2^/ mice were transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes (Figure 6). These findings also support the hypothesis that CD4⁺ CD25⁺ T cells enhance the Th2-type immune response. However, because it has been shown that antigen-specific IgE antibody is not essential for the airway eosinophilia in a murine model of asthma (27), the decreased IgE production would not account for the diminished eosinophil recruitment into the airways in Rag-2^/ mice that were transferred with CD4⁺ CD25⁺ T cell-depleted splenocytes.

In contrast to the functional properties of CD4⁺ CD25⁺ T cells, the development of CD4⁺ CD25⁺ T cells is still largely unknown. It has been shown that CD4⁺ CD25⁺ T cells are present in the thymus and these cells exhibit functional properties similar to those of peripheral CD4⁺ CD25⁺ T cells (28, 29). Moreover, thymectomy at 3 d of life decreases CD4⁺ CD25⁺ T cells from the periphery (8), suggesting that the origin of CD4⁺ CD25⁺ T cells is in the thymus.

Interestingly, the development of CD4⁺ CD25⁺ T cells is IL-2 dependent (28), whereas IL-2 is not essential for the development of thymocytes and peripheral CD4⁺ CD25 T cells (30). Moreover, we previously showed that CD4⁺ CD25⁺ T cells were absent in mice lacking _c (31), an essential receptor component for IL-2, IL-7, IL-9, and IL-15 signaling (23). These findings indicate that IL-2 signaling via _c is essential for the development of CD4⁺ CD25⁺ T cells. Among intracellular signaling molecules under _c, Stat5a and Stat5b are known to potently regulate the expression of CD25 by directly binding to the 5' regulatory region of the CD25 gene (32, 33). Therefore, we examined the development of CD4⁺ CD25⁺ T cells in DO10 Stat5a^/ mice and found that the number of CD4⁺ CD25⁺ T cells was severely decreased in DO10 Stat5a^/ mice (34). This observation may account for our previous findings that anti-CD3-stimulated splenocytes in Stat5a^/ mice produced less IL-4 and IL-5 than wild-type mice and that antigen-induced eosinophil recruitment into the airways was decreased in Stat5a^/ mice (21).

In addition to IL-2 signaling, signaling from TCRs (29) as well as costimulatory molecules (16) is essential for the development of CD4⁺ CD25⁺ T cells. DO10 mice in Rag-2-deficient background lack CD4⁺ CD25⁺ T cells (29 and our unpublished data), suggesting that the expression of endogenous TCRs and their ligation are essential for the development of CD4⁺ CD25⁺ T cells. Moreover, Salomon and coworkers (16) have demonstrated that the CD28/B7 pathway is also essential for the development of CD4⁺ CD25⁺ T cells. Thus, these observations imply that all of the _c-dependent signaling, TCR signaling, and CD28 signaling are essential for the development of CD4⁺ CD25⁺ T cells in vivo. In contrast, the development of CD4⁺ CD25⁺ T cells is normal in mice lacking IL-4 or Stat6 (our unpublished data), indicating that the Th2 environment is not essential for the development of CD4⁺ CD25⁺ T cells.

In summary, we have shown that CD4⁺ CD25⁺ T cells modulate the helper T cell differentiation toward the Th2 type and thus upregulate Th2 cell-mediated allergic inflammation in the airways. These results suggest that antagonism of CD4⁺ CD25⁺ T cells would be a rational therapeutic approach to allergic inflammation such as asthma.