October 2006
Volume 47, Issue 10
Free
Immunology and Microbiology  |   October 2006
CD4+PD-1+ T Cells Acting as Regulatory Cells during the Induction of Anterior Chamber-Associated Immune Deviation
Author Affiliations
  • Qianli Meng
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Peizeng Yang
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Bing Li
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Hongyan Zhou
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Xiangkun Huang
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Lianxiang Zhu
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Yalin Ren
    From the Zhongshan Ophthalmic Center, Uveitis Study Center, Sun Yat-sen University, Key Laboratory of Ophthalmology, Ministry of Education, Guangzhou, People’s Republic of China; and the
  • Aize Kijlstra
    Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, The Netherlands.
Investigative Ophthalmology & Visual Science October 2006, Vol.47, 4444-4452. doi:10.1167/iovs.06-0201
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      Qianli Meng, Peizeng Yang, Bing Li, Hongyan Zhou, Xiangkun Huang, Lianxiang Zhu, Yalin Ren, Aize Kijlstra; CD4+PD-1+ T Cells Acting as Regulatory Cells during the Induction of Anterior Chamber-Associated Immune Deviation. Invest. Ophthalmol. Vis. Sci. 2006;47(10):4444-4452. doi: 10.1167/iovs.06-0201.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To study the expression and functional characteristics of programmed death-1 (PD-1) and its ligands in the spleens of mice undergoing anterior chamber–associated immune deviation (ACAID).

methods. ACAID was induced in BALB/c mice by intracameral injection of ovalbumin (OVA). The expression of PD-1 and its ligands in the spleens of ACAID mice was determined by quantitative real-time PCR, Western blotting, and flow cytometry. In vitro proliferation assays, enzyme-linked immunosorbent assays, and adoptive transfer assays were used to investigate the functional characteristics of splenic CD4+PD-1+ T cells of ACAID mice.

results. Both mRNA and protein of PD-1, PD-L1, and PD-L2 were markedly upregulated in the spleens of ACAID mice compared with controls. CD4+PD-1+ T cells from ACAID mice produced large amounts of IL-10 and exhibited in vitro antigen-specific suppressive activity. CD4+PD-1+ T cells from ACAID mice were able to significantly inhibit the antigen-specific, delayed-type hypersensitivity response when adoptively transferred to naive mice.

conclusions. CD4+PD-1+ T cells from ACAID mice, as regulatory cells, are involved in the induction of antigen-specific suppression in association with enhanced expression of IL-10. CD4+PD-1+ T cells in the murine spleen may represent a substantial population of regulatory T cells possibly responsible for the induction of ACAID after intracameral injection of antigen.

Injection of soluble antigen (Ag) into the anterior chamber (AC) of the eye induces systemic tolerance known as anterior chamber–associated immune deviation (ACAID). 1 2 3 4 It is characterized by an Ag-specific inhibition of the delayed-type hypersensitivity (DTH) response and a reduction in complement-fixing antibodies (Abs). 5 The mechanisms involved in the induction of ACAID have been extensively studied during recent years. Previous studies have demonstrated that T cells, 6 7 an intact eye for 3 days after injection, 8 9 the spleen, 10 and the ocular immunosuppressive microenvironment 11 12 are required for the tolerance induction in ACAID. Studies by Wilbanks et al. 13 have provided evidence that at least two functionally distinct regulatory T (Treg) cell populations are involved in the ACAID response. One Treg cell population phenotypically belonging to the CD4+ T-cell subpopulation inhibits the induction of DTH (an afferent regulator). The other Treg cell population is composed of CD8+ T cells capable of inhibiting the expression of DTH (an efferent regulator). 
An immune response is regulated through different mechanisms, 14 including regulatory costimulatory molecules. 15 The balance between the positive and negative signaling costimulatory pathways dictates the fate of individual T cells and the immune response. As a member of the CD28/B7 family and costimulatory molecules, PD-1 interacts with its ligands and constitutes a recently discovered inhibitory regulatory pathway. 16 17 18 PD-1 was originally identified by Ishida et al. 19 as a molecule linked to in vitro induction of apoptotic cell death in murine lymphoid cell lines. PD-1 contains an immunoreceptor tyrosine–based inhibiting motif (ITIM) and an immunoreceptor tyrosine–based switch motif (ITSM) 20 and is transcriptionally induced in activated T cells, B cells, and myeloid cells, suggesting that it has broader roles in immune regulation. 21 22 The ligands for PD-1 (PD-Ls) are PD-L1 and PD-L2, also known as B7-H1 and B7-DC. PD-L1 is constitutively expressed in T cells, B cells, macrophages, dendritic cells (DCs) and nonlymphoid cells, whereas PD-L2 is expressed restrictedly in activated macrophages and DCs. 23 24 25 26 Previous studies have demonstrated that PD-1−/− C57BL/6 mice develop lupuslike arthritis and glomerulonephritis 27 and that PD-1−/− BALB/c mice develop autoantibody-mediated dilated cardiomyopathy. 28 Blockade of the PD-1 ligand during experimental autoimmune encephalomyelitis or diabetes was shown to exacerbate these diseases. 29 30 Polymorphisms in the human PD-1 gene (PDCD1) are associated with the development of systemic lupus erythematosus, rheumatoid arthritis, diabetes, and multiple sclerosis. 31 32 33 34 Like CTLA-4, the ligation of PD-1 on T cells suppresses T-cell proliferation and cytokine production. 35 36 Moreover, this pathway has been implicated in blocking allograft rejection, modulating T- and B-cell–dependent pathologic immune responses, and inducing transplantation tolerance. 16 37 38 All the results emphasize the importance of the PD-1/PD-L pathway in down-modulating immune responses and in inducing and maintaining peripheral tolerance. 
Given the importance of PD-1 as an immunosuppressive molecule, we hypothesized that it could also be involved in the induction of Treg cells in ACAID. In this study, we showed that PD-1 and its ligands were upregulated in the spleens of ACAID mice. The CD4+PD-1+ T cells from ACAID mice exhibited an Ag-specific suppressive role in cell proliferation and DTH response in association with an increased secretion of IL-10. These results indicate that CD4+PD-1+ T cells may function as Treg cells in the development of ACAID and that PD-1 expression may be a marker distinguishing subpopulations of Treg cells. 
Methods
Animals
Female BALB/c mice, 6 to 8 weeks of age, were purchased from the animal facility at Sun Yat-sen University (GuangZhou, China). All mice were kept under specific pathogen-free conditions and were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Induction of ACAID
ACAID was induced as described previously using microinjection of Ag into the AC of the eye of BALB/c mice. 39 Briefly, the mice were anesthetized with inhalation anesthesia consisting of oxygen and 1.7% isoflurane. A glass micropipette (approximately 80 μm in diameter) was fitted onto a sterile infant feeding tube and mounted onto a 0.1-mL Hamilton syringe. A Hamilton automatic dispensing apparatus was used to inject 2 μL of the 20 mg/mL ovalbumin (OVA; grade V, EC no. 232-692-7; Sigma, St. Louis, MO) solution, dissolved in phosphate-buffered saline (PBS), into the AC of the right eye. Seven days later, the mice were immunized by subcutaneous injection of 250 μg OVA in 100 μL PBS emulsified 1:1 in complete Freund adjuvant (CFA; Sigma). Each animal received a total volume of 200 μL. Mice receiving subcutaneous injections of OVA in CFA alone were used as positive controls (PCs). Mice receiving intracameral injection of 5 μL sterile PBS alone were used as negative controls (NCs). 
DTH Assay
Seven days after subcutaneous immunization, mice were challenged by intradermal injection of 200 μg OVA in 20 μL PBS into the right ear pinnae. The left ear pinnae received 20 μL sterile PBS alone. Both ear pinnae were measured with an engineer’s micrometer (Mitutoyo, Kanagawa, Japan) before and 24 hours after OVA challenge, and the difference in ear pinnae size was used as a measurement of DTH. Results were expressed as: specific ear pinnae swelling = (24-h measurement – 0-h measurement) of right ear – (24-h measurement – 0-h measurement) of left ear. 
Expression of PD-1 and PD-1 Ligand mRNA Assayed by Fluorescent Quantitative Real-Time PCR
For detecting the expression of PD-1 and PD-1 ligands, total RNA was extracted from the spleens of the experimental and control mice using reagent (TRIzol; Invitrogen, Carlsbad, CA) and was quantified by absorbance at 260 nm. Single-strand cDNA was synthesized with an RT-PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The reaction mixture was incubated at 37°C for 60 minutes and then by 95°C for 3 minutes. Real-time PCR was performed in a sequence detection system (PE Prism 7000; Perkin-Elmer Applied Biosystems, Foster City, CA). Sequences of the forward (FW) and reverse (RV) primers and probes (TP) for PD-1, PD-L1, and PD-L2 were as follows: PD-1-FW, 5′-CCGCCTTCTGTAATGGTTTGA-3′; PD-1-RV, 5′-GGGCAGCTGTATGATCTGGAA-3′; PD-1-TP, 5′-FAM-AACCCGTCCAGGATGCCCG-TAMRA-3′; PD-L1-FW, 5′-TGGACAAACAGTGACCACCAA-3′; PD-L1-RV, 5′-CCCCTCTGTCCGGGAAGT-3′; PD-L1-TP, 5′-FAM-CCGTGAGTGGGAAGAGAAGTGTCAC-TAMRA-3′; PD-L2-FW, 5′-GCCCCTGGGAAAGGCTTT-3′; PD-L2-RV, 5′-CGGTACTGCCCGGAATCTC-3′; PD-L2-TP, 5′-FAM-TTCCACATCCCTAGTGTCCAAGTG-TAMRA-3′. The optimized protocol included an initial denaturation step of 93°C for 120 seconds, followed by 40 cycles of 93°C for 45 seconds and 55°C for 60 seconds. PD-1, PD-L1, and PD-L2 mRNA levels were expressed as copy number per microgram RNA after normalization with different concentrations of the PCR products of the PD-1, PD-L1, and PD-L2 genes. Three mice were used in each group, and experiments were repeated at least three times. 
Expression of PD-1 and PD-1 Ligand Protein Assayed by Western Blotting
Total protein was extracted from the obtained spleens with the mammalian cell lysis kit (Bio Basic; East Markham Ontario, Canada). Samples were centrifuged at 100,000g for 45 minutes, and supernatants were collected and stored at –70°C. Protein in each sample was quantified by the conventional Bradford method. Samples containing equal amounts of protein (35 μg protein/lane) were separated on 10% SDS-PAGE, followed by transfer onto polyvinylidene fluoride (PVDF) membranes (Boehringer Mannheim, Mannheim, Germany). After membranes were blocked with PBS containing 3% skimmed milk for 1 hour at room temperature, they were incubated with rat anti–mouse PD-1 (1:200; BioLegend, San Diego, CA), goat anti–mouse PD-L1 (1:500; R&D Systems, Minneapolis, MN), or rat anti–mouse PD-L2 (1:200; BioLegend) monoclonal antibody (mAb) in PBS at 4°C overnight. Membranes were washed three times with TBST and subsequently incubated with horseradish peroxidase–conjugated anti–rat IgG or anti–goat IgG (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. Finally, the membrane was washed three times with TBST, and the resultant signal was detected by enhanced chemiluminescence with a reagent kit (LumiGLO; Cell Signaling Technology, Beverly, MA). To quantitate the samples, the chemiluminescent-stained polyvinylidene difluoride (PVDF) membrane was quenched in stripped solution for 30 minutes at 50°C. After blocking, the membrane was soaked in PBS containing mouse anti–mouse actin (1:400; NeoMarkers, Fremont, CA) and subsequently was incubated with horseradish peroxidase–conjugated anti–mouse IgG (1:2000; Santa Cruz Biotechnology). The computer-aided one-dimensional gel analysis program was used to quantitate the signals. Three mice were used in each group, and experiments were repeated at least three times. 
Phenotype of PD-1 Detected by Flow Cytometry Analysis
The obtained spleens were pressed through nylon mesh to produce a single-cell suspension. Red blood cells were lysed with Tris-NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Cells (1 × 106) were stained with rat anti–mouse FITC-conjugated anti–CD3 mAb (BioLegend), APC-conjugated anti–CD4 mAb (BioLegend), or PE-conjugated anti–PD-1 mAb (BioLegend) for 30 minutes at 4°C in the dark. PE-conjugated rat IgG2b (BioLegend) served as isotype control. Cells were washed twice in 1% bovine serum albumin (BSA)/PBS and were analyzed (FACSAria; BD Biosciences, San Jose, CA) with a software program (FACSDiVa; BD Biosciences). 
Magnetic Affinity Cell Sorting
CD4+ T cells were purified from the splenocytes of BALB/c mice using a CD4+ T-cell isolation kit (negative selection) (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Retained cells were eluted from the column and shown by flow cytometry (FCM) to be enriched CD4 T cells (purity greater than 95%). Flow-through cells—enriched CD4+ T cells of purity greater than 90%—were then stained with PE-conjugated anti–mouse PD-1 mAb (BioLegend) for 30 minutes at 4°C in the dark followed by anti–PE microbeads (Miltenyi Biotec) for 15 minutes at 4°C. Cells were washed with 0.5% BSA in PBS followed by magnetic separation using MS+ columns as described by the manufacturer. Retained cells were eluted from the column and shown to be enriched CD4+PD-1+ T cells (purity greater than 90%). Flow-through cells were enriched CD4+PD-1 T cells (purity greater than 90%). 
In Vitro Proliferation Assay and Cytokine Assay by Enzyme-Linked Immunosorbent Assays
CD4 cells, acting as APCs, were prepared from splenocytes of naive BALB/c mice according to the method described and were treated with 50 μg/mL mitomycin C (MMC; Sigma) for 45 minutes at 37°C. CD4+PD-1 (1 × 105 cells/well), CD4+PD-1+ (1 × 105 cells/well) T cells from naive, PC, and ACAID mice were cultured with MMC-treated CD4 cells (5 × 105 cells/well) in complete medium (RPMI 1640 medium [Gibco] containing 10% heat-inactivated fetal bovine serum [FBS], 50 μM 2-mercaptoethanol, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 1% streptomycin/penicillin) in 96-well, round-bottom plates, and were supplemented with 1 μg/mL anti–CD3 mAb (145 to 2C11; eBioscience) at 37°C in 5% CO2. Purified CD4+PD-1 T cells (1 × 105 cells/well, as responder cells) from naive, PC, and ACAID groups were cocultured with different concentrations of purified CD4+PD-1+ T cells (as suppressor cells) from the corresponding groups and MMC-treated CD4 T cells (5 × 105 cells/well, as APCs) in complete medium in 96-well, round-bottom plates and were stimulated with 100 μg/mL OVA at 37°C in 5% CO2. The ratio of CD4+PD-1 T cells to CD4+PD-1+ T cells was 1:0, 1:0.25, 1:0.5, or 1:1. To determine proliferation rates, each well was pulsed with 1 μCi [3H] thymidine (Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) for the last 9 hours of a 72-hour culture and was subsequently measured by scintillation counter (LS6500; Beckman Coulter, Hialeah, CA). To calculate the suppression percentage, the cpm value from each group was divided by the average cpm value without Treg cells. Results in the experiments without stimulation of anti–CD3 mAb or OVA were also analyzed. 
To determine IL-10 production, freshly isolated splenic CD4+PD-1+ T cells from mice with naive, PC, or ACAID were cultured with MMC-treated CD4 T cells in complete medium in 96-well, round-bottom plates and were stimulated with 100 μg/mL OVA for 48 hours. An experiment without OVA stimulation was used as control. Supernatants were collected and assayed by ELISA (Biosource International, Camarillo, CA) according to the manufacturer’s instructions. Sensitivity and range of the assay were 13 pg/mL and 31.2 to 2000 pg/mL, respectively. 
Adoptive Transfer
To assess the regulatory effect and the Ag specificity of CD4+PD-1+ T cells, an adoptive transfer experiment was performed. In brief, CD4+PD-1+ and CD4+PD-1 T cells from splenocytes of ACAID mice were sorted as described and were injected intravenously into the tail veins of naive BALB/c mice at a concentration of 1 × 106 cells/200 μL or 5 × 106 cells/200 μL. Three days later, 250 μg OVA or BSA in CFA was injected subcutaneously. DTH assay was performed 7 days after subcutaneous immunization. Three mice were used in each group, and each experiment was repeated at least three times. 
Statistical Analysis
Results were expressed as mean ± SD. Data were subjected to analysis by ANOVA (SPSS 11.0; SPSS, Chicago, IL). P < 0.05 was considered significantly different. 
Results
Upregulated Expression of PD-1, PD-L1, and PD-L2 in the Spleens of ACAID Mice
We investigated the expression of PD-1, PD-L1, and PD-L2 by inducing ACAID through intracameral injection with OVA and collecting spleens for analysis of mRNA expression. mRNA of PD-1, PD-L1, and PD-L2 was detectable in all groups through fluorescent quantitative (FQ) real-time PCR, but it was significantly higher in ACAID mice than in naive, NC, and PC groups (PD-1: P < 0.001 vs. naive, NC, and PC; PD-L1: P = 0.003 vs. PC or P < 0.001 vs. naive and NC; PD-L2: P = 0.001 vs. PC or P < 0.001 vs. naive and NC; Fig. 1 ). mRNA expression of PD-1, PD-L1, and PD-L2 was also increased in the PC group but did not reach significant difference compared with the naive and NC groups. 
To examine the protein expression of PD-1 and its ligands, we performed Western blot analysis in the spleens of naive, NC, PC, and ACAID mice. A protein with a molecular weight of approximately 55 kDa and corresponding to the reported size for PD-1 was detected at a low level in the spleens of naive and NC mice. PD-1 protein expression was increased in PC mice and significantly upregulated in ACAID mice. Proteins with a molecular weight of approximately 43 kDa or 42 kDa, respectively, for PD-L1 or PD-L2 were also detected in the spleens of all groups. Similarly, the protein expression for PD-L1 and PD-L2 in ACAID mice was significantly higher than in the naive, NC, and PC groups (PD-1: P = 0.046 [PC], P = 0.021 [naive], P = 0.015 [NC]; PD-L1: P = 0.049 [PC], P = 0.036 [naive], P = 0.035 [NC]; PD-L2: P = 0.044 [PC], P = 0.024 [naive], P = 0.027 [NC]; Fig. 2 ). 
Increased Frequency of Splenic CD4+PD-1+ T Cells in ACAID Mice
In view of the results of upregulation of mRNA and protein of PD-1 in the spleens of ACAID mice, we further examined the expression of PD-1 on splenic T lymphocytes by FCM. Three-color FCM analysis of splenocytes from naive, NC, PC, or ACAID mice was performed using mAb against CD3, CD4, or PD-1. The results showed that PD-1 was expressed on a small fraction of CD4+ and CD4 (CD8+) T cells in all groups. Statistical analysis showed a significantly higher frequency of CD4+PD-1+ T cells than of CD8+PD-1+ T cells in each group (P < 0.001, 0.001, P = 0.002, 0.008 for CD4+PD-1+ T cells vs. CD8+PD-1+ T cells in naive, NC, PC, or ACAID groups, respectively; Fig. 3 ). In addition, the frequency of CD4+PD-1+ T cells was significantly higher in ACAID mice than in the other three groups (P < 0.001). The PC mice also showed an increased frequency of CD4+PD-1+ T cells compared with naive and NC mice, though the difference was not significant. Similarly, an increased frequency of CD8+PD-1+ T cells was observed in ACAID mice compared with naive, NC, and PC mice (P < 0.001 [naive], P = 0.002 [NC], and P = 0.004 [PC] for CD8+PD-1+ T cells). 
Ag-Specific Suppressive Activity of CD4+PD-1+ T Cells from ACAID Mice In Vitro
To characterize the CD4+PD-1+ T cell functions in vitro, we isolated CD4+PD-1 T cells and CD4+PD-1+ T cells from splenocytes by magnetically activated cell sorter (MACS; Fig. 4A ). The CD4+PD-1+ T cells from each group showed weak proliferation in response to anti–CD3 mAb stimulation in the presence of MMC-treated CD4 splenocytes as APCs, whereas CD4+PD-1 T cells from each group showed vigorous proliferation. Control experiments without anti–CD3 mAb stimulation showed that CD4+PD-1+ T cells and CD4+PD-1+ T cells displayed weak proliferation. 
To examine the feature of Ag specificity, a hallmark of the ACAID phenomenon, we further studied the suppressive activity of CD4+PD-1+ T cells when exposed to OVA stimulation. As shown in Figure 4B , the CD4+PD-1+ T cells from ACAID mice showed a significantly stronger suppressive effect on the proliferation of CD4+PD-1 T cells than those from PC groups, and this suppression was expressed in a dose-dependent manner. CD4+PD-1 T cells from naive mice responded at a very low level on stimulation with OVA. The inhibition of CD4+PD-1+ T cells from naive mice on the CD4+PD-1 T cells was not obviously observed in the experiment with coculture of these two subsets because of the much lower basal proliferation of CD4+PD-1 T cells. The experiment without OVA stimulation revealed that CD4+PD-1 T cells from each group showed weak proliferation, and CD4+PD-1+ T cells did not show suppression on the proliferation of CD4+PD-1 T cells. 
Because IL-10 was considered necessary for the generation of ACAID, 39 40 our study further examined whether CD4+PD-1+ T cells in ACAID produce IL-10. We used ELISA to measure this cytokine in the supernatants of the cultures described. All CD4+PD-1+ T cells from naive, PC, and ACAID groups, when stimulated with OVA, produced IL-10. Notably, the CD4+PD-1+ T cells from ACAID mice produced a higher level of IL-10 than those from the other controls (P = 0.032). The experiment without OVA stimulation showed that the level of IL-10 secreted by CD4+PD-1+ T cells from each group was below the detection limit of the assay (Fig. 4C)
Adoptively Transferred Tolerance by CD4+PD-1+ T Cells from ACAID Mice
Given that tolerance induced by intracameral injection of Ag can be adoptively transferred by CD4+ cells isolated from the spleens of ACAID mice, 13 our study tested whether CD4+PD-1+ T cells from mice with ACAID could transfer the impaired DTH response. As shown in Figure 5 , when CD4+PD-1+ T cells were transferred, DTH responses to OVA, but not to BSA, were markedly suppressed (P < 0.001). However, a vigorous DTH response was detected when CD4+PD-1 T cells were transferred at both 1 × 106 and 5 × 106 cells. 
Discussion
Recent studies have shown that the PD-1/PD-L pathway has a crucial role in the maintenance of immunologic privilege in the murine and the human placentas. 41 42 Our study examined whether this pathway could also play a role in the development of ACAID. Our results revealed that mRNA and protein of PD-1, PD-L1, and PD-L2 were significantly increased in the spleens of ACAID mice. CD4+PD-1+ T cells showed an increased secretion of IL-10 from ACAID mice and were more effective in suppressing the proliferation of CD4+PD-1 T cells than those from the control groups used in our experiments. CD4+PD-1+ T cells from ACAID mice were able to significantly inhibit the Ag-specific DTH response when adoptively transferred to naive mice. All these results suggest that CD4+PD-1+ T cells, as substantial Treg cells, are involved in the induction of ACAID and that PD-1 seems to act as one of the markers that can identify subpopulations of Treg cells. 
Because the spleen is the central processing unit for tolerance induction in ACAID, 10 43 44 we first investigated the expressions of PD-1 and its ligands at the mRNA and protein levels in this tissue. These results showed that PD-1 and its ligands, PD-L1 and PD-L2, were significantly upregulated in ACAID mice and that intracameral injection of PBS did not influence the expression of these molecules. Our FCM analysis of mice only receiving intracameral injection of OVA did not show a different frequency of CD4+PD-1+ T cells in the spleen (data not shown) compared with naive mice. This result indicates that intracamerally injected Ag by itself could not initiate a change detectable by the technique used in this study. It has been reported that CFA can promote T-cell activation and that the activated T cells may upregulate PD-1 and PD-Ls expression. 21 45 Therefore, the observed twofold to threefold increases in RNA and protein expression for PD-1 and its ligands from ACAID mice represent the sum of Ag-specific and nonspecific response. Further experiments using mice that received another irrelevant Ag injected into the AC before challenge with OVA in CFA may clarify this issue. In contrast to our results, Liang et al. 23 did not detect protein expression of PD-L2 in the spleens of naive BALB/c mice using immunofluorescence. This difference may be a result of the sensitivity of the techniques used in our study. 
PD-1 is known to be expressed on activated T cells and to be involved in the downregulation of T-cell activation. 45 PD-1 has been shown to inhibit the proliferation and cytokine production of murine CD4+ and CD8+ T cells 36 and to play a critical role in the regulation of CD4 and CD8 alloimmune responses. 38 Phenotypic analysis suggests that CD4+PD-1+ T cells producing large amounts of IL-10 constitute a unique anergic T-cell subset in rheumatoid arthritis synovial fluid. 32 Therefore, we considered it important to phenotypically identify the PD-1+ T cells in the splenocytes during ACAID. Our study revealed that the expression of PD-1 on CD4+ T cells was significantly higher than that on CD8+ T cells in each group, suggesting that CD4+PD-1+ T cells possibly have a stronger immune regulatory function than CD8+PD-1+ T cells. Consequently, we focused primarily on CD4+PD-1+ T cells in the subsequent experiments. 
It has been reported that CD4+ T cells isolated from the spleens of ACAID mice can downregulate T-cell proliferative responses to specific Ag and produce large amounts of IL-10. 13 40 46 IL-10 mRNA and protein are upregulated during the induction of systemic tolerance after Ag inoculation into the eye, and its production is crucial for the induction of ACAID. 39 40 47 Our in vitro study showed that CD4+PD-1+ T cells from ACAID mice were hypoproliferative, produced large amounts of IL-10, and inhibited the proliferation of CD4+PD-1 T cells on stimulation with the priming antigen (OVA). Our in vivo experiment showed an Ag-specific inhibition of CD4+PD-1+ T cells on the DTH response when these cells were adoptively transferred into naive mice. It is likely that these CD4+PD-1+ T cells from ACAID mice exerted regulatory activities, thereby contributing to the induction of ACAID. Our finding is consistent with the result that a subset of PD-1+ T cells could downregulate T cell responses and have suppressive properties. 36 48 Unlike the CD4+PD-1+ T cells, CD4+PD-1 T cells from ACAID mice did not show a suppressive role in DTH responses even when the amount of transferred cells was increased fivefold. It seems that CD4+PD-1 T cells do not have, or have only very weak, regulatory function. Another possibility may be that these cells act as mediators of inflammatory or autoimmune disease. Further study is needed to characterize this subset during ACAID. 
Recent studies have revealed that PD-1 mRNA is expressed at high levels in CD4+CD25+ Treg cells and anergic T cells. 49 50 Totsuka et al. 48 have identified two subpopulations of Treg cells expressing PD-1 in the spleens of naive mice—CD4+CD25+PD-1+ T cells and CD4+CD25PD-1+ T cells. Both subpopulations exhibited suppressive activity in vitro and prevented CD4+CD45RBhigh T-cell–induced colitis, suggesting that they have a potent regulatory role. Our study focused on the CD4+PD-1+ T cells and also showed a potent regulatory role of these cells in the ACAID model. All these results indicate that CD4+PD-1+ T cells in the murine spleen may, at least in part, contribute to the suppressive activity of CD4+CD25+ Treg cells and represent a substantial population of Treg cells possibly responsible for the induction of ACAID. 
It has been demonstrated that PD-1 regulates T-cell homeostasis in the periphery. 16 17 18 22 However, the functions of its ligands, PD-L1 and PD-L2, are still controversial. A number of studies have shown that the interaction of PD-1 with PD-L1 or PD-L2 could inhibit T-cell receptor–mediated T-cell proliferation and cytokine production. 19 21 Other studies have suggested that PD-L1 and PD-L2 may costimulate T cells after interaction with an unidentified receptor. 51 52 The present study showed a concurrently increased expression of PD-1 and both its ligands in the spleens of mice undergoing ACAID. These results suggest that PD-1, perhaps not the uncertain receptor, interacts with PD-L1 or PD-L2, thereby contributing to the development of ACAID. However, it is also necessary to identify other receptors for PD-L1 and PD-L2 and to investigate the role of each in ACAID. 
In conclusion, our results suggest that CD4+PD-1+ T cells from ACAID mice represent an important subset of regulatory cells, as evidenced by their suppressive role in cell proliferation, production of a large amount of IL-10, and adoptive transfer of Ag-specific immune tolerance to naive mice. However, our studies have some limitations. We still do not know the unambiguous phenotype and profile of cytokines of CD4+PD-1+ T cells or the mechanisms involved in their immunosuppressive properties. The expression of PD-1 and PD-Ls in the murine eye, one of the target organs of ACAID, should also be studied. Experiments with PD-1 or PD-L knockout mice or with anti PD-1 antibodies to block the PD-1/PD-L pathway will greatly help us to understand the role of PD-1 in the induction of ACAID and may substantially contribute to the study on the strategies to prevent and treat autoimmune diseases such as uveitis. 
 
Figure 1.
 
Expression of PD-1, PD-L1, and PD-L2 mRNA in the spleen was determined by FQ real-time PCR. Expression of PD-1 (A), PD-L1 (B), or PD-L2 (C) in spleens from naive, NC, PC, or ACAID mice was shown as copy number per microgram RNA after normalization with different PCR products of the target gene. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. Data represent the mean ± SD of three independent experiments. *P < 0.001 and **P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 1.
 
Expression of PD-1, PD-L1, and PD-L2 mRNA in the spleen was determined by FQ real-time PCR. Expression of PD-1 (A), PD-L1 (B), or PD-L2 (C) in spleens from naive, NC, PC, or ACAID mice was shown as copy number per microgram RNA after normalization with different PCR products of the target gene. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. Data represent the mean ± SD of three independent experiments. *P < 0.001 and **P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 2.
 
Expression of PD-1, PD-L1, and PD-L2 protein in the spleen was determined by Western blotting. (A) Equal amounts of protein (35 μg) were loaded onto each lane. Bands of 55 kDa, 43 kDa, and 42 kDa corresponding to the reported sizes of PD-1, PD-L1, and PD-L2, respectively, were detected. A representative blot of three separate experiments is shown. (B) Quantitative analysis of PD-1, PD-L1, and PD-L2 protein in each group is shown using computer-aided one-dimensional gel analysis. Data represent the mean ± SD of three independent experiments. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *#†P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 2.
 
Expression of PD-1, PD-L1, and PD-L2 protein in the spleen was determined by Western blotting. (A) Equal amounts of protein (35 μg) were loaded onto each lane. Bands of 55 kDa, 43 kDa, and 42 kDa corresponding to the reported sizes of PD-1, PD-L1, and PD-L2, respectively, were detected. A representative blot of three separate experiments is shown. (B) Quantitative analysis of PD-1, PD-L1, and PD-L2 protein in each group is shown using computer-aided one-dimensional gel analysis. Data represent the mean ± SD of three independent experiments. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *#†P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 3.
 
Expression of PD-1 on murine splenic T lymphocytes was analyzed by FCM. Splenocytes were prepared and labeled with anti–CD3+, anti–CD4+, and anti–PD-1+ mAb. (A) CD3+, CD3+CD4+, and CD3+CD4 populations were gated and analyzed for the expression of PD-1+ cells. Gray lines represent staining with isotype control IgG. Colored lines represent staining with anti–PD-1 mAb. Percentage of PD-1–positive cells is indicated. Results of a representative experiment are shown. (B) FCM histogram showed the expression of PD-1 on murine splenic T lymphocytes. All data shown represent the mean ± SD of at least three independent experiments. Mice that received intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PC mice were immunized subcutaneously with OVA in CFA. NC mice received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *P < 0.05 for the frequency of PD-1+ cells on CD3+CD4+ compared with those on CD3+CD4 in each group. #P < 0.001 for the frequency of PD-1+ cells on CD3+CD4+ in ACAID groups compared with those in naive, NC, or PC groups. †P < 0.05 for the frequency of PD-1+ cells on CD3+CD4 in ACAID groups compared with those in naive, NC, or PC groups.
Figure 3.
 
Expression of PD-1 on murine splenic T lymphocytes was analyzed by FCM. Splenocytes were prepared and labeled with anti–CD3+, anti–CD4+, and anti–PD-1+ mAb. (A) CD3+, CD3+CD4+, and CD3+CD4 populations were gated and analyzed for the expression of PD-1+ cells. Gray lines represent staining with isotype control IgG. Colored lines represent staining with anti–PD-1 mAb. Percentage of PD-1–positive cells is indicated. Results of a representative experiment are shown. (B) FCM histogram showed the expression of PD-1 on murine splenic T lymphocytes. All data shown represent the mean ± SD of at least three independent experiments. Mice that received intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PC mice were immunized subcutaneously with OVA in CFA. NC mice received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *P < 0.05 for the frequency of PD-1+ cells on CD3+CD4+ compared with those on CD3+CD4 in each group. #P < 0.001 for the frequency of PD-1+ cells on CD3+CD4+ in ACAID groups compared with those in naive, NC, or PC groups. †P < 0.05 for the frequency of PD-1+ cells on CD3+CD4 in ACAID groups compared with those in naive, NC, or PC groups.
Figure 4.
 
CD4+PD-1+ T cells from ACAID mice showed Ag-specific suppressive activity in vitro. (A) Splenic CD4+PD-1+ T cells were isolated by MACS. Left: FCM shows CD4+PD-1+ and CD4+PD-1 T cells before selection. Middle: FCM shows the enriched CD4+PD-1+ T cells tagged with PE-conjugated anti–mouse PD-1 mAb and anti–PE microbeads. Right: FCM shows that the flow-through cells are enriched CD4+PD-1 T cells. Numbers represent the purity of the CD4+PD-1+ or the CD4+PD-1 population. Typical profiles of the sorted fractions are shown. (B) Suppressive effect of CD4+PD-1+ T cells on the proliferation of CD4+PD-1 T cells. Purified CD4+PD-1 T cells (1 × 105 cells/well, as responder cells) from naive, PC, or ACAID groups were cocultured with purified CD4+PD-1+ T cells (as suppressor cells) from the corresponding groups at different responder/suppressor ratios (1:0, 1:0.25, 1:0.5, and 1:1) in the presence of MMC-treated CD4 T cells (5 × 105 cells/well, as APCs) and OVA (100 μg/mL) for 72 hours. [3H] thymidine was added for the last 9 hours and measured by scintillation counter. Baseline cpm (no suppressors) for the PC group with or without OVA stimulation was 2300 or 910, respectively. Data are represented as mean ± SD of triplicate samples. *P < 0.05 for ACAID compared with PC groups at different responder/suppressor ratios. (C) IL-10 production. CD4+PD-1+ T cells from mice with naive, PC, or ACAID were stimulated with or without OVA (100 μg/mL) in the presence of APCs for 48 hours. IL-10 in the supernatants measured by ELISA. Data are represented as the mean ± SD of triplicate samples. *P < 0.05 for ACAID groups compared with PC groups. §IL-10 produced by CD4+PD-1+ T in the absence of OVA was lower than the detection limit of the assay.
Figure 4.
 
CD4+PD-1+ T cells from ACAID mice showed Ag-specific suppressive activity in vitro. (A) Splenic CD4+PD-1+ T cells were isolated by MACS. Left: FCM shows CD4+PD-1+ and CD4+PD-1 T cells before selection. Middle: FCM shows the enriched CD4+PD-1+ T cells tagged with PE-conjugated anti–mouse PD-1 mAb and anti–PE microbeads. Right: FCM shows that the flow-through cells are enriched CD4+PD-1 T cells. Numbers represent the purity of the CD4+PD-1+ or the CD4+PD-1 population. Typical profiles of the sorted fractions are shown. (B) Suppressive effect of CD4+PD-1+ T cells on the proliferation of CD4+PD-1 T cells. Purified CD4+PD-1 T cells (1 × 105 cells/well, as responder cells) from naive, PC, or ACAID groups were cocultured with purified CD4+PD-1+ T cells (as suppressor cells) from the corresponding groups at different responder/suppressor ratios (1:0, 1:0.25, 1:0.5, and 1:1) in the presence of MMC-treated CD4 T cells (5 × 105 cells/well, as APCs) and OVA (100 μg/mL) for 72 hours. [3H] thymidine was added for the last 9 hours and measured by scintillation counter. Baseline cpm (no suppressors) for the PC group with or without OVA stimulation was 2300 or 910, respectively. Data are represented as mean ± SD of triplicate samples. *P < 0.05 for ACAID compared with PC groups at different responder/suppressor ratios. (C) IL-10 production. CD4+PD-1+ T cells from mice with naive, PC, or ACAID were stimulated with or without OVA (100 μg/mL) in the presence of APCs for 48 hours. IL-10 in the supernatants measured by ELISA. Data are represented as the mean ± SD of triplicate samples. *P < 0.05 for ACAID groups compared with PC groups. §IL-10 produced by CD4+PD-1+ T in the absence of OVA was lower than the detection limit of the assay.
Figure 5.
 
CD4+PD-1+ T cells from ACAID mice could induce downregulation of OVA-specific DTH. Splenic CD4+PD-1+ and CD4+PD-1 T cells from ACAID mice were sorted and injected intravenously into naive BALB/c mice. Three days later, 250 μg OVA or BSA in CFA was injected subcutaneously. DTH assay was performed 7 days after subcutaneous immunization. Mice were injected intravenously with CD4+PD-1+ T cells (1 × 106) or CD4+PD-1 T cells (1 × 106 or 5 × 106) from ACAID mice, immunized subcutaneously with OVA or BSA in CFA, and underwent ear challenge as experimental groups. Mice that received ear challenge (OVA or BSA) alone served as NCs. Mice were immunized subcutaneously with OVA or BSA in CFA and underwent ear challenge as PCs. All data shown represent the mean ± SD of at least three independent experiments (n = 3). *Mean results significantly lower than those of PC (P < 0.001).
Figure 5.
 
CD4+PD-1+ T cells from ACAID mice could induce downregulation of OVA-specific DTH. Splenic CD4+PD-1+ and CD4+PD-1 T cells from ACAID mice were sorted and injected intravenously into naive BALB/c mice. Three days later, 250 μg OVA or BSA in CFA was injected subcutaneously. DTH assay was performed 7 days after subcutaneous immunization. Mice were injected intravenously with CD4+PD-1+ T cells (1 × 106) or CD4+PD-1 T cells (1 × 106 or 5 × 106) from ACAID mice, immunized subcutaneously with OVA or BSA in CFA, and underwent ear challenge as experimental groups. Mice that received ear challenge (OVA or BSA) alone served as NCs. Mice were immunized subcutaneously with OVA or BSA in CFA and underwent ear challenge as PCs. All data shown represent the mean ± SD of at least three independent experiments (n = 3). *Mean results significantly lower than those of PC (P < 0.001).
The authors thank Changyou Wu for his constructive suggestions and Haining Zhang for her technical assistance. 
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Figure 1.
 
Expression of PD-1, PD-L1, and PD-L2 mRNA in the spleen was determined by FQ real-time PCR. Expression of PD-1 (A), PD-L1 (B), or PD-L2 (C) in spleens from naive, NC, PC, or ACAID mice was shown as copy number per microgram RNA after normalization with different PCR products of the target gene. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. Data represent the mean ± SD of three independent experiments. *P < 0.001 and **P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 1.
 
Expression of PD-1, PD-L1, and PD-L2 mRNA in the spleen was determined by FQ real-time PCR. Expression of PD-1 (A), PD-L1 (B), or PD-L2 (C) in spleens from naive, NC, PC, or ACAID mice was shown as copy number per microgram RNA after normalization with different PCR products of the target gene. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. Data represent the mean ± SD of three independent experiments. *P < 0.001 and **P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 2.
 
Expression of PD-1, PD-L1, and PD-L2 protein in the spleen was determined by Western blotting. (A) Equal amounts of protein (35 μg) were loaded onto each lane. Bands of 55 kDa, 43 kDa, and 42 kDa corresponding to the reported sizes of PD-1, PD-L1, and PD-L2, respectively, were detected. A representative blot of three separate experiments is shown. (B) Quantitative analysis of PD-1, PD-L1, and PD-L2 protein in each group is shown using computer-aided one-dimensional gel analysis. Data represent the mean ± SD of three independent experiments. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *#†P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 2.
 
Expression of PD-1, PD-L1, and PD-L2 protein in the spleen was determined by Western blotting. (A) Equal amounts of protein (35 μg) were loaded onto each lane. Bands of 55 kDa, 43 kDa, and 42 kDa corresponding to the reported sizes of PD-1, PD-L1, and PD-L2, respectively, were detected. A representative blot of three separate experiments is shown. (B) Quantitative analysis of PD-1, PD-L1, and PD-L2 protein in each group is shown using computer-aided one-dimensional gel analysis. Data represent the mean ± SD of three independent experiments. Mice receiving intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PCs were immunized subcutaneously with OVA in CFA. NCs received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *#†P < 0.05 for ACAID groups versus naive, NC, and PC groups.
Figure 3.
 
Expression of PD-1 on murine splenic T lymphocytes was analyzed by FCM. Splenocytes were prepared and labeled with anti–CD3+, anti–CD4+, and anti–PD-1+ mAb. (A) CD3+, CD3+CD4+, and CD3+CD4 populations were gated and analyzed for the expression of PD-1+ cells. Gray lines represent staining with isotype control IgG. Colored lines represent staining with anti–PD-1 mAb. Percentage of PD-1–positive cells is indicated. Results of a representative experiment are shown. (B) FCM histogram showed the expression of PD-1 on murine splenic T lymphocytes. All data shown represent the mean ± SD of at least three independent experiments. Mice that received intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PC mice were immunized subcutaneously with OVA in CFA. NC mice received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *P < 0.05 for the frequency of PD-1+ cells on CD3+CD4+ compared with those on CD3+CD4 in each group. #P < 0.001 for the frequency of PD-1+ cells on CD3+CD4+ in ACAID groups compared with those in naive, NC, or PC groups. †P < 0.05 for the frequency of PD-1+ cells on CD3+CD4 in ACAID groups compared with those in naive, NC, or PC groups.
Figure 3.
 
Expression of PD-1 on murine splenic T lymphocytes was analyzed by FCM. Splenocytes were prepared and labeled with anti–CD3+, anti–CD4+, and anti–PD-1+ mAb. (A) CD3+, CD3+CD4+, and CD3+CD4 populations were gated and analyzed for the expression of PD-1+ cells. Gray lines represent staining with isotype control IgG. Colored lines represent staining with anti–PD-1 mAb. Percentage of PD-1–positive cells is indicated. Results of a representative experiment are shown. (B) FCM histogram showed the expression of PD-1 on murine splenic T lymphocytes. All data shown represent the mean ± SD of at least three independent experiments. Mice that received intracameral injection of OVA followed by subcutaneous injection with OVA and CFA after 7 days acted as ACAID mice. PC mice were immunized subcutaneously with OVA in CFA. NC mice received intraocular injection of PBS alone. All spleens from each group were harvested on day 7 after the last treatment. *P < 0.05 for the frequency of PD-1+ cells on CD3+CD4+ compared with those on CD3+CD4 in each group. #P < 0.001 for the frequency of PD-1+ cells on CD3+CD4+ in ACAID groups compared with those in naive, NC, or PC groups. †P < 0.05 for the frequency of PD-1+ cells on CD3+CD4 in ACAID groups compared with those in naive, NC, or PC groups.
Figure 4.
 
CD4+PD-1+ T cells from ACAID mice showed Ag-specific suppressive activity in vitro. (A) Splenic CD4+PD-1+ T cells were isolated by MACS. Left: FCM shows CD4+PD-1+ and CD4+PD-1 T cells before selection. Middle: FCM shows the enriched CD4+PD-1+ T cells tagged with PE-conjugated anti–mouse PD-1 mAb and anti–PE microbeads. Right: FCM shows that the flow-through cells are enriched CD4+PD-1 T cells. Numbers represent the purity of the CD4+PD-1+ or the CD4+PD-1 population. Typical profiles of the sorted fractions are shown. (B) Suppressive effect of CD4+PD-1+ T cells on the proliferation of CD4+PD-1 T cells. Purified CD4+PD-1 T cells (1 × 105 cells/well, as responder cells) from naive, PC, or ACAID groups were cocultured with purified CD4+PD-1+ T cells (as suppressor cells) from the corresponding groups at different responder/suppressor ratios (1:0, 1:0.25, 1:0.5, and 1:1) in the presence of MMC-treated CD4 T cells (5 × 105 cells/well, as APCs) and OVA (100 μg/mL) for 72 hours. [3H] thymidine was added for the last 9 hours and measured by scintillation counter. Baseline cpm (no suppressors) for the PC group with or without OVA stimulation was 2300 or 910, respectively. Data are represented as mean ± SD of triplicate samples. *P < 0.05 for ACAID compared with PC groups at different responder/suppressor ratios. (C) IL-10 production. CD4+PD-1+ T cells from mice with naive, PC, or ACAID were stimulated with or without OVA (100 μg/mL) in the presence of APCs for 48 hours. IL-10 in the supernatants measured by ELISA. Data are represented as the mean ± SD of triplicate samples. *P < 0.05 for ACAID groups compared with PC groups. §IL-10 produced by CD4+PD-1+ T in the absence of OVA was lower than the detection limit of the assay.
Figure 4.
 
CD4+PD-1+ T cells from ACAID mice showed Ag-specific suppressive activity in vitro. (A) Splenic CD4+PD-1+ T cells were isolated by MACS. Left: FCM shows CD4+PD-1+ and CD4+PD-1 T cells before selection. Middle: FCM shows the enriched CD4+PD-1+ T cells tagged with PE-conjugated anti–mouse PD-1 mAb and anti–PE microbeads. Right: FCM shows that the flow-through cells are enriched CD4+PD-1 T cells. Numbers represent the purity of the CD4+PD-1+ or the CD4+PD-1 population. Typical profiles of the sorted fractions are shown. (B) Suppressive effect of CD4+PD-1+ T cells on the proliferation of CD4+PD-1 T cells. Purified CD4+PD-1 T cells (1 × 105 cells/well, as responder cells) from naive, PC, or ACAID groups were cocultured with purified CD4+PD-1+ T cells (as suppressor cells) from the corresponding groups at different responder/suppressor ratios (1:0, 1:0.25, 1:0.5, and 1:1) in the presence of MMC-treated CD4 T cells (5 × 105 cells/well, as APCs) and OVA (100 μg/mL) for 72 hours. [3H] thymidine was added for the last 9 hours and measured by scintillation counter. Baseline cpm (no suppressors) for the PC group with or without OVA stimulation was 2300 or 910, respectively. Data are represented as mean ± SD of triplicate samples. *P < 0.05 for ACAID compared with PC groups at different responder/suppressor ratios. (C) IL-10 production. CD4+PD-1+ T cells from mice with naive, PC, or ACAID were stimulated with or without OVA (100 μg/mL) in the presence of APCs for 48 hours. IL-10 in the supernatants measured by ELISA. Data are represented as the mean ± SD of triplicate samples. *P < 0.05 for ACAID groups compared with PC groups. §IL-10 produced by CD4+PD-1+ T in the absence of OVA was lower than the detection limit of the assay.
Figure 5.
 
CD4+PD-1+ T cells from ACAID mice could induce downregulation of OVA-specific DTH. Splenic CD4+PD-1+ and CD4+PD-1 T cells from ACAID mice were sorted and injected intravenously into naive BALB/c mice. Three days later, 250 μg OVA or BSA in CFA was injected subcutaneously. DTH assay was performed 7 days after subcutaneous immunization. Mice were injected intravenously with CD4+PD-1+ T cells (1 × 106) or CD4+PD-1 T cells (1 × 106 or 5 × 106) from ACAID mice, immunized subcutaneously with OVA or BSA in CFA, and underwent ear challenge as experimental groups. Mice that received ear challenge (OVA or BSA) alone served as NCs. Mice were immunized subcutaneously with OVA or BSA in CFA and underwent ear challenge as PCs. All data shown represent the mean ± SD of at least three independent experiments (n = 3). *Mean results significantly lower than those of PC (P < 0.001).
Figure 5.
 
CD4+PD-1+ T cells from ACAID mice could induce downregulation of OVA-specific DTH. Splenic CD4+PD-1+ and CD4+PD-1 T cells from ACAID mice were sorted and injected intravenously into naive BALB/c mice. Three days later, 250 μg OVA or BSA in CFA was injected subcutaneously. DTH assay was performed 7 days after subcutaneous immunization. Mice were injected intravenously with CD4+PD-1+ T cells (1 × 106) or CD4+PD-1 T cells (1 × 106 or 5 × 106) from ACAID mice, immunized subcutaneously with OVA or BSA in CFA, and underwent ear challenge as experimental groups. Mice that received ear challenge (OVA or BSA) alone served as NCs. Mice were immunized subcutaneously with OVA or BSA in CFA and underwent ear challenge as PCs. All data shown represent the mean ± SD of at least three independent experiments (n = 3). *Mean results significantly lower than those of PC (P < 0.001).
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