CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification

Cellular Immunology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm

CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification Dongwei Chen a,1, Ying Wang a,1, Huan Wang a, Yiqing Wu a, Sheng Xia b,⇑, Minghui Zhang a,⇑ a b

Institute of Immunology, Medical Center, Tsinghua University, Beijing, People’s Republic of China Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 5 December 2014 Revised 9 May 2015 Accepted 9 May 2015 Available online xxxx Keywords: CD8+ T cell Dendritic cell CD4+ T cell Immune tolerance Asthma

a b s t r a c t Emerging evidence has suggested that CD8+ T had modulatory function on CD4+ T mediated autoimmune and inflammatory diseases. However, the underlying mechanisms remain unclear. In this study, we found that CD8+ T activation inhibited OVA323–339 antigen specific CD4+ T cells proliferation in vitro and in vivo. Further investigation demonstrated that this immunosuppression largely depended on the soluble factor from activated CD8+ T to modify the phenotype and functions of DCs. Moreover, not only the inhibitors for IDO or iNOS, but also IFN-c neutralization markedly reversed this immunosuppression on OVA323–339 antigen specific CD4+ T cells proliferation. Interestingly, CD8+ T cells absence aggravated the pathological damage in lung in OVA-induced asthma model, but alleviated by CD8+ T transfer and activation. Thus, these findings suggested that activated CD8+ T population exerted feedback regulation in DCs modification, and then attenuated CD4+ T mediated immune response. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction As one system with complicated biological structures and processes, the immune system relies on sophisticated regulatory networks to maintain homeostasis. In recent decades, a variety of immunoregulatory factors have been widely reported, especially the immunomodulatory functions of regulatory CD4+ T cells. Comparatively, the counterparts of CD8+ T cells are less defined but attracting increasing attention. CD8+ T cells are generally recognized as a cytotoxic T-cell population, which plays a critical role in anti-viral infection, tumor cell elimination, and allograft rejection. In contrast, accumulating CD8+ Abbreviations: 1-MT, 1-methyl-tryptophan; BAL, bronchoalveolar lavage; CFSE, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester; CMFDA, 5-chloromethylfluorescein diacetate; DC, dendritic cell; Far-Red, CELLTRACE(TM) FAR RED DDAO-SE; HE, hematoxylin and eosin; IDO, indoleamine-pyrrole 2,3-dioxygenase; IFN-c, interferon-c; IL, interleukin; iNOS, inducible nitric oxide synthase; OVApep-loaded DCs, OVA323–339 + OVA257–264-loaded DCs; PAS, periodic acid-Schiff; PBIT, S,S0 -(1,3-phenylenebis[1,2-ethanediyl]) bisisothiourea; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SN, supernatant isolated from the activated CD8+ T cells; SN-modified DCs, supernatant-modified DCs; TGF-b, transforming growth factor-b. ⇑ Corresponding authors. Tel.: +86 (511) 8503 8449; fax: +86 (511) 8503 8483 (S. Xia). Tel.: +86 (10) 6279 2592; fax: +86 (10) 6279 0232 (M. Zhang). E-mail addresses: [email protected] (S. Xia), [email protected] (M. Zhang). 1 Dongwei Chen and Ying Wang contribute equally to this manuscript.

T subsets with immunoregulatory/immunosuppressive capacities have been reported in CD4+ T-associated autoimmune and inflammatory diseases [1], including asthma [2], systemic lupus erythematosus (SLE) [3], inflammatory bowel disease (IBD) [4,5], and experimental autoimmune encephalomyelitis (EAE) [6,7]. Several potential mechanisms for the immunoregulation of these CD8+ T on CD4+ T-mediated immunity have been explored, such as immune regulatory cytokines secretion (e.g., IL-10, TGF-b, IFN-c) [8], cell–cell direct contact [9,10], and cytotoxic lysis of APCs in a perforin-dependent or Fas-dependent manner [11,12]. Collectively, these data suggested that CD8+ T could exert regulatory functions in immunity through multiple mechanisms. As a type of professional antigen presenting cells, dendritic cells (DCs) play pivotal roles in CD4+ T activation and proliferation. The phenotypes and functions of these cells are highly plastic and modifiable by factors in the ambient environment [13,14]. Our and other’s studies have shown that some special microenvironments can drive mature and immature DCs to differentiate into regulatory DCs, thereby suppressing their ability to induce CD4+ T responses [15–18]. Activated CD8+ T cells express many adhesion molecules on the cell membrane, and secret different cytokine profiles from its quiescent state. These adhesion molecules and cytokines work as indispensable mediators involving in the cross-talk of immune cells in immune response. Therefore, in a similar fashion, activated CD8+ T cells might generate a special immune microenvironment, and indicated their potential regulatory functions on DCs, and then

http://dx.doi.org/10.1016/j.cellimm.2015.05.001 0008-8749/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

2

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

ultimately on the activation and proliferation of CD4+ T cells. But, up to now, the exact roles of activated CD8+ T in immune regulation are not well demonstrated. In this study, we explored the functions of activated CD8+ T in immune regulation by using T-cell/DC co-culture in vitro and CD8+ T adoptive transfer and activation in vivo. Indeed, we found that CD8+ T activation negatively regulated CD4+ T cells proliferation through altering the phenotypes and functions of DCs. Thus, our findings provided a new clue to clarify the crosstalk between T cells and DCs, and indicated the potential regulatory roles of activated CD8+ T in allergic airway inflammation.

the method described previously [15]. To prepare OVA loaded DCs, maDCs were incubated with 400 ng/mL OVA257–264 and/or 1 lg/mL OVA323–339 peptides for 24 h. After residual free peptides were removed, peptide-loaded DCs were resuspended in PBS and then were infused i.v. at a dose of 1.5  106 per mouse 24 h after T cells being transferred. To prepare supernatant modified DCs, OT-1 CD8+ T (1  106) co-cultured with maDCs (1  105) for 36 or 60 h, and supernatant (SN) was collected. Supernatant modified DCs (SN-modified DCs) were prepared by incubating OVA loaded maDCs with activated CD8+ T derived supernatant and fresh RPMI-1640 complete medium (1:1, v:v) for 48 h. In some experiments, CD11c+ splenic DC cells were used.

2. Materials and methods 2.5. Flow cytometry analysis 2.1. Ethics statement All experimental protocols were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Tsinghua University, Beijing, China. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering. 2.2. Mice All mice were bred and maintained in specific, pathogen-free conditions at the experimental animal platform of Tsinghua University, and were used at 6–8 weeks of age. C57BL/6 and BALB/c mice were obtained from Chinese Academy of Medical Sciences (CAMS). OT-1 transgenic (Tg) mice [19], and OT-2 Tg mice [20] were provided by Charles River Laboratories (Wilmington, MA, USA). b2m null mice, which are deficient in MHC class I protein expression and have few CD8+ T cells [21]; DO11.10 mice (BALB/c background) carrying Tg CD4 TCR specific for H2Kd-OVA323–339 residues [20]; IFN-cR(/) mice on C57BL/6 genetic background [22]; and C.Cg-Foxp3tm2Tch/J mice on BALB/c genetic background (in which 97% of Foxp3+ T cells are identifiable by EGFP+) were all purchased from the Jackson Laboratory (Bar Harbor, ME, USA). 2.3. Reagents All cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin, and 100 lg/mL streptomycin. RPMI 1640 medium and fetal calf serum (FCS) were purchased from PAA Laboratories (Cölbe, Germany). Recombinant murine cytokines GM-CSF, IL-4, IL-6, IFN-c were purchased from ProsPec (Otisville, NY, USA). Fluorescein-conjugated monoclonal antibodies and isotype control antibodies were purchased from Biolegend (San Diego, CA, USA). Neutralizing antibodies to IFN-c and TGF-b were purchased from R&D Systems (Minneapolis, MN, USA). Anti-CD4, CD8 and CD11c magnetic microbeads were provided by Miltenyi Biotec (Bergisch Gladbach, Germany). Primary antibodies to iNOS and pSTAT3 were purchased from Cell Signaling Technology (Beverly, MA, USA), anti-IL-10 and anti-TGF-b were from R&D Systems, IDO antibody was purchased from Biolegend, and anti-IL-12p40 was provided by eBioscience (San Diego, CA, USA). CFSE, CMFDA, Far-Red and propidium iodide (PI) were obtained from Invitrogen (Carlsbad, CA, USA). Ovalbumin (OVA, grade V), OVA257–264 (OT-1 peptide), OVA323–339, PBIT and 1-MT were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.4. Dendritic cells (DCs) preparation Bone Marrow derived mature DCs (maDCs) were prepared from C57BL/6, BALB/c or IFN-cR(-/-) mouse bone marrow according to

The phenotype of immune cells, phagocytic ability of maDCs and T cells proliferation were assayed by using flow cytometry (FCM) (FACSAria II, B.D. Biosciences) as described previously [15]. The data were analyzed using FlowJo software (TreeStar Inc.; Ashland, OR, USA). 2.6. The purification and proliferation assays of T cells CD4+ and CD8+ T cells were purified by positive selection with magnetic-activated cell sorting (MACS). The purity of cells was routinely more than 90%. For CD4+ T proliferation assay in vitro, 2  105 CFSE-labeled OT-2 CD4+ T cells were co-cultured with 2  104 OVApep-loaded maDCs in the presence or absence of 1  105 OT-1 CD8+ T cells in triplicate in 96-well round-bottom plates. The divisions of CD4+ T were assessed 72 or 96 h later by using FCM analysis. In some experiments, IDO inhibitor 1-MT (200 lM), iNOS inhibitor PBIT (10 lM) or TGFb neutralizing antibodies (10 lg/mL) were added. For CD4+ T proliferation assay in vivo, 4  106 CFSE-labeled OT-2 CD4+ T cells were transferred i.v. with or without 2  106 OT-1 CD8+ T cells into C57BL/6 recipient 24 h prior to 1.5  106 peptide-loaded maDCs challenge. Four days later, the percentage of CFSE+CD4+ T cells in total live CD4+ T in the spleen and blood was assessed by FCM. In some experiments, CD4+ T cells from DO11.10  C57BL/6 F1 hybrid mice were transferred i.v. into BALB/c  C57BL/6 F1 hybrid mice 24 h prior to peptide-loaded DCs injection, which were treated by activated CD8+ T cells supernatant. Four days after DCs challenge, the proliferation of CD4+ T was assessed by determining the percentages of KJ1-26+CD4+ T cells in total live CD4+ T cells in the spleen and blood sample in recipient using FCM. 2.7. Foxp3+ Treg differentiation assay in vitro CD4+ T-cell and CD8+ T-cell were isolated from DO11.10  Foxp3EGFP F1 hybrid mice and OT-1  Balb/C F1 hybrid mice, respectively. CD4+ T cells were co-cultured in the presence or absence of CD8+ T-cell with OVApep-loaded maDCs for 48 h, and then were collected to detect the frequency of CD4+CD25+Foxp3+ T cells (Treg cells) among total CD4+ T cells. 2.8. Cytokines profile analysis OT-1 CD8+ T (2  106) and OVA257–264-loaded maDCs (2  105) were co-cultured for 36 h or 60 h in 24-well plates (2 mL per well). Supernatants were collected, and frozen at 80 °C for further assay. The levels of cytokines in supernatants were analyzed using a mouse Th1/Th2/Th17/Th22 13plex flowcytomix assay kit (eBioscience; San Diego, CA, USA) on the FACSAria II (BD Biosciences; San Jose, CA, USA). Flowcytomix™ Pro Software was used for data acquisition and analysis.

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

2.9. Absolute cell number counted by flow cytometer

3. Results

To count the absolute cell number of each well in 96-well plate, microspheres (1  105, 3.27 lm in diameter) were firstly added into wells as internal control. Then, cells were collected and stained with fluorescent labeled antibodies. After being re-suspended in an exact volume of 150 lL PBS, cells were collected by using flow cytometer (FACSAria II, BD Biosciences). For each sample, 5  104 microspheres were acquired and facilitated the calculation of absolute numbers of target cells (CD4+ T cells or CMFDA+ PI DCs).

3.1. Activated CD8+ T suppressed CD4+ T cells proliferation

2.10. Western blot analysis Western blotting was done as previous reported [17]. In brief, protein was extracted from DCs by lysis buffer and boiled for 10 min. Then, the samples were separated by SDS–PAGE and transferred by electroblotting to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with antibodies specific for IDO, IL-10, IL-12p40, iNOS, TGF-b, pSTAT3 and GAPDH, and further assayed by horseradish peroxidase (HRP)-labeled antibody. Multiple exposures on Kodak films (Eastman Kodak; Rochester, NY) were performed, and the visualized data were scanned with a scanning apparatus (BENQ Corp.; Taipei, Taiwan). 2.11. OVA-induced asthma and CD8+ T-cell intervention To prepare asthma in mice, C57BL/6 WT mice and b2m null mice were sensitized on day 0 by intraperitoneal injection (i.p.) of 10 lg of chicken OVA (grade V) in 0.5 mL phosphate-buffered saline (PBS) containing 20 mg of aluminum hydroxide adjuvant. These mice were further re-challenged with OVA (i.p) on days 7 and 14. Next, CD8+ OT-1 T cells (4  106) together with 2  106 OVA257–264-loaded maDCs were infused intravenously (i.v.) on day 23. To induce asthma, from day 24 to day 28, these mice were daily exposed to aerosolized MOG or OVA (1% w/v) for 30 min. After the last aerosol challenge, blood was collected from the orbital sinus, and then mice were sacrificed for further experiments. To prepare broncho-alveolar lavage (BAL) fluids, the airways and lungs were flushed with 800 lL of cold PBS containing 0.1% BSA and 0.05 lM EDTA for three times, and BAL fluids were used immediately or frozen at 80 °C for experiments. Both the total numbers of white blood cells (WBC) in BAL fluids and the absolute numbers of differential immune cells were counted by an automated hematology analyzer (Beckman Coulter; Brea, CA, USA). In some experiments, OVA loaded DCs (2  106) were infused i.n. respectively and OVA inhalational challenges were performed on days 14 and 19. The levels of IL-4 and IL-5 in BAL fluid were detected using a flowcytomix assay kit (eBioscience; San Diego, CA, USA). 2.12. Histology of lung Lung tissue blocks were collected and embedded with O.T.C. for quick freezing. Tissue sections (5 lm) were sliced and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) for histological examination. The histological images were obtained using a microscope (Nikon; Tokyo, Japan) equipped with a digital camera (Canon; Tokyo, Japan). 2.13. Statistical analysis Data were from one representative of at least 3 independent experiments, and were depicted as the mean ± SEM. Statistical analysis was performed using two-tailed Student’s t-test for two-group comparison. And p value < 0.05 was considered as statistical significance.

3

To explore the direct effects of activated CD8+ T on CD4+ T cells proliferation, we cultured OVA323–339 (OT-2) specific CD4+ T cells in the presence or absence of OVA257–264 (OT-1) specific CD8+ T cells together with OVA323–339 + OVA257–264-loaded DCs (OVApep-loaded DCs) in vitro. In contrast to the substantial proliferation of DC-primed OVA323–339 specific CD4+ T cells, the presence of CD8+ T obviously suppressed the divisions of CD4+ T cells after being cultured for three days in vitro (Fig. 1A). This CD8+ T mediated inhibition on CD4+ T cells was CD8+ T/CD4+ T ratio dependent (Fig. 1B). To further evaluate this immune suppression in vivo, we transferred CFSE-labeled OT-2 CD4+ T cells with or without OT-1 CD8+ T into C57BL/6 recipients, and then intravenously challenged with OVApep-loaded maDCs 24 h later. The data in Fig. 1C showed that OVA257–264 specific OT-1 CD8+ T transfer and activation significantly reduced the frequency of OVA323–339 specific CFSE+CD4+ T cells among total CD4+ T in blood and spleen in recipient mice in vivo. These data demonstrated that OVA257–264 specific CD8+ T activation had the capability in inhibiting OVA323–339 antigen specific CD4+ T cells proliferation in vitro and in vivo. 3.2. Activated CD8+ T had no effect on CD4+ T activation and Treg generation As the CD4+ T proliferation was not only tightly associated with its differentiation, but also with its activation, we next want to know if the immune regulation of activated CD8+ T via regulating the activation of CD4+ T cells. By detecting the expressions of activation markers CD25, CD69 and CD44 on CD4+ T cells, the results in Fig. 2A shown that activated CD8+ T had no obviously suppressive effects on maDC-induced CD4+ T activation in vitro. Regulatory T cells (Tregs), which can be identified by their expression of CD4, CD25 and Forkhead box P3 (Foxp3), showed suppressive capabilities on CD4+ T activation and proliferation [23,24]. We further analyzed the effects of activated CD8+ T on Treg differentiation, and the data showed no significant difference in the frequencies of Tregs between groups with and without activated CD8+ T (Fig. 2B). Thus, these results suggested that the immune regulation of activated CD8+ T on CD4+ T cells proliferation depended neither on CD4+ T activation nor on CD4+ Tregs generation. 3.3. Activated CD8+ T modified DCs through soluble factors To further explore whether activated CD8+ T meditated immune suppression was dependent on either CD4+ T/CD8+ T direct interaction or an APC-dependent manner, maDCs were fixed by polyformaldehyde to eliminate any APC cell modification. As we described previously [15], fixed maDCs partly maintained their ability to prime CD4+ T cells proliferation. The data in Fig. 3A showed that CD8+ T activation had no obvious inhibitory effects on fixed maDC-primed CD4+ T cells proliferation (Fig. 3A), and suggested that DC modification might contribute to the immune suppression of activated CD8+ T. Interestingly, activated CD8+ T had no obvious cytotoxicity on maDCs, even at different dose of antigen peptides (ranging from 0 to 1 lg/mL) and different CD8+ T/maDC ratios (5:1, 10:1, 20:1) (Fig. 3B). The data in Fig. 3C also showed that activated CD8+ T modified DCs showed stronger phagocytotic activity. Furthermore, we did ex vivo experiment to detect if activated CD8+ T had similar effects on different CD11c+ splenic DC subsets. The results in Fig. 3D showed that the expressions of CD86 and MHC II in all three splenic DC subsets (CD8a+ DCs, CD4+ DCs and CD8CD4 DCs) were down regulated, and indicated

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

4

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

Fig. 1. CD8+ T activation suppressed OVA-specific CD4+ T cells proliferation in vitro and in vivo. (A) CD4+ T cells proliferation inhibited by activated CD8+ T in vitro. CFSElabeled OT-2 CD4+ T cells (2  105) co-cultured with maDCs (2  104) loaded with OVApep in U-bottom 96-well plates in the presence or absence of OT-1 CD8+ T cells (1  105). Cell divisions of live CD4+ T were analyzed by detecting CFSE dye dilution with FACSAria after 72 h culture. Representative flow cytometry (FCM) histograms were shown; the numbers in graphs were the percentages of divided CD4+ T in total live CD4+ T cells. (B) The suppressive function of activated CD8+ T was in a dose-dependent manner. The cells were treated as (A), but with different CD8+ T/CD4+ T ratios. The proliferation of CFSE-labeled OT-2 CD4+ T cells was assayed by FCM. (C) CD4+ T cells proliferation was inhibited by adoptively transferred and activated CD8+ T in vivo. Recipient C57BL/6 mice were transferred i.v. 4  106 CFSE-labeled OT-2 CD4+ T cells in the presence or absence of 2  106 OT-1 CD8+ T, and were challenged i.v. with 1.5  106 OVApep-loaded maDCs 24 h later. Blood samples and spleens were collected to prepare single-cell suspensions 96 h after maDCs were transferred, and the percentages of CFSE+CD4+ T cells in total CD4+ T in blood and spleens were analyzed by FCM. Data in graphs were displayed as mean ± SEM (n = 5). **p < 0.01; OVApep-loaded maDCs, maDCs were loaded with OVA257–264 + OVA323–339. Data represent one of three experiments with similar results.

that activated CD8+ T had negative regulatory on the phenotypes of splenic DCs. As the membrane proteins or soluble factors may do contribution in activated CD8+ T-induced DC modification, we next separated CD8+ T from DC cells using transwell co-culture. The results in Fig. 3E showed that activated CD8+ T kept its capability in suppressing OVA-specific CD4+ T cells proliferation even if these cells were separated from maDC by transwell. To identify the exact effects of soluble factors from activated CD8+ T on maDC modification, maDCs were cultured with supernatant (SN) isolated from the activated CD8+ T to prepare supernatant-modified DCs (SN-modified DCs), and its functions and phenotypes were further analyzed. The results showed SN-modified DCs had less efficient ability than untreated DCs in priming antigen specific CD4+ T cells proliferation in vitro (Fig. 4A), which also was confirmed by T cells adaptive transfer experiment in vivo (Fig. 4B). Furthermore, SN-modified DCs primed CD4+ T cells produced lower levels of IL-2 and IFN-c than unmodified DCs cohorts (Fig. 4C). Similarly, the capability of SN-modified DCs in priming OVA257–264 CD8+ T proliferation was also been inhibited (Fig. 4D). Thus, these results indicated that not only direct cell–cell contact between activated CD8+ T and maDCs, but also soluble factors from activated CD8+ T contributed to maDC functions modification. Next, we detected the phenotypes of DCs, which treated with activated CD8+ T derived supernatant. The data in Fig. 4E showed that the expression levels of CD11c, as well as the co-stimulatory molecules (CD80/CD86) and I-Ab on both CD8+ T-reacted DCs and SN-modified DCs were significantly decreased. Instead, B7-H1, an immune negative regulator [25], was increased in these DCs comparing to control maDCs (Fig. 4E). Thus, the phenotype of these

activated CD8+ T modified DCs displayed the characteristics of tolerogenic DCs as previously reported [15]. Furthermore, it had been reported that the functions of tolerogenic DCs were closely related to inducible nitric oxide synthase (iNOS), indoleamine 2,3-dioxygenase (IDO) and immunosuppressive cytokines, such as TGF-b and IL-10 [13]. Indeed, the western blot data in Fig. 4E showed that the levels of iNOS, IDO and TGF-b were increased in SN-modified DCs, but no difference was founded in IL-10 and IL-12 (Fig. 4F). Interestingly, NO synthase inhibitor (PBIT) and IDO inhibitor (1-MT) could partly restore the efficiency of SN-modified DCs mediated OVA323–339 antigen specific CD4+ T cells proliferation, but not found in TGF-b blocking wells (Fig. 4G). As previously reported, the phosphorylation of signal transducers and activators of transcription 3 (STAT 3) in DCs was associated with the expression of nitric oxide, TGF-b and IDO [26,27], we next detected the levels of pSTAT3 in DCs, and found more pSTAT3 could be detected in SN-modified DCs (Fig. 4E). Collectively, these data suggested that activated CD8+ T-derived soluble factors modified maDCs with tolerogenic DCs properties. 3.4. IFN-c worked as the maDC modification contributor To explore which soluble factor contributes to maDC modification, the levels of different cytokines (IL-2, IL-4, IL-6, IL-17, IFN-c and TNF-a) in the supernatant of activated CD8+ T were quantified with flowcytomic assay. The results indicated that the highest was IFN-c, and then followed by IL-6 and IL-2 (Fig. 5A). Considering the role of IFN-c in immunoregulation [28] and its substantial secretion from activated CD8+ T cells, we assumed that IFN-c may play an essential function in activated CD8+ T mediated immune

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

Fig. 2. Activated CD8+ T exerted no effects on CD4+ T activation and Treg generation. (A) The effect of activated CD8+ T on maDC-induced antigen specific CD4+ T activation. After being cultured with OT-1 CD8+ T cells and OVA-loaded maDCs (CD4+ T:CD8+ T:maDCs = 10:5/0:1) for 72 h, the expression of CD25, CD69 and CD44 on OT-2 CD4+ T cells were detected by FCM. Representative FCM graphs were shown. The number in histogram indicates the representative mean fluorescence intensity (MFI) of CD4+ T; shadow graph was isotype control. (B) The effect of activated CD8+ T on the generation of CD4+CD25+FoxP3+ Tregs. OVA323–339 specific CD4+ T cells from DO11.10  Foxp3EGFP F1 mice were cultured for 48 h as described in (A), and cells were collected to detect the percentage of CD25+Foxp3+ CD4+ cells in total CD4+ T. Representative graphs were shown; numbers in the graphs represent the percentages of CD4+ Treg. The data were from triplicate wells in one representative experiment and shown as mean ± SEM. These experiments were performed at least three times.

suppression on CD4+ T cells. Indeed, exogenous IFN-c significantly inhibited OVA323–339 antigen specific CD4+ T cells proliferation (Fig. 5B). In contrast, exogenous IL-6 had not shown any effects (Fig. 5B). To further demonstrate the direct function of IFN-c on DCs modification, we used polyformaldehyde-fixed DCs or IFN-cR(/) maDCs to prime CD4+ T into activation and proliferation. The results showed that exogenous IFN-c failed to suppress the proliferation of CD4+ T cells, which primed by fixed or IFN-cR deficient maDCs, even its concentration up to 1000 ng/mL (Fig. 5C and D). But, if the supernatant from activated CD8+ T was pretreated with IFN-c neutralizing monoclonal antibody, its immunosuppressive capability was partially reversed (Fig. 5E). Collectively, these data suggested that the IFN-c, derived from activated CD8+ T, contributed to maDC modification and then suppressed antigen specific CD4+ T cells proliferation. 3.5. Activated CD8+ T alleviated OVA-specific CD4+ T mediated asthma Given the fact that the airway inflammation in allergic asthma were primarily mediated by allergen-primed CD4+ T activation

5

and proliferation, we further analyzed the exact role of activated CD8+ T in asthma by using CD8+ T deficient b2m null mice. After the mice were sensitized with OVA/aluminum hydroxide 3 times, and repeatedly challenged with inhaled OVA for 5 consecutive days (Fig. 6A), the numbers of bronchoalveolar lavage (BAL) cells increased significantly in CD8+ T deficient mice (Fig. 6B). By analyzing the composition of the BALs, we further found that the majority of the infiltrated cells in b2m null mice were eosinophils and monocytes/macrophages (Fig. 6B). Interestingly, adoptive transfer and activation of OVA257–264 specific CD8+ T before exposure to aerosolized OVA obviously alleviated the recruitment of eosinophils and monocytes/macrophages in bronchia alveolus (Fig. 6B). These results were further confirmed by the histological data in Fig. 6D. Comparing with wild type mice, severe inflammatory cell infiltration in pericapillaries and peribronchial area, and more goblet cell metaplasia can be found in b2m null asthmatic mice (Fig. 6D). If these mice were pre-treated by activated CD8+ T, the inflammation and tissue damage in lung would be largely relieved (Fig. 6D). It has been shown that a large release of IL-4, IL-5 and IL-13 plays important roles in asthma development [29,30]. Therefore, we analyzed the levels of IL-4, IL-5 and IL-13 in BAL fluid, and found that the levels of these cytokines were dramatically increased in CD8+ T deficient mice, but were suppressed by CD8+ T transfer and activation (Fig. 6C). While, CD8+ T cells transfer increased the secretion of IFN-c in BAL fluid both in WT and b2m null asthmatic mice (Fig. 6C). Thus, these results suggested that the activation of CD8+ T displayed protective effects against airway inflammation in OVA-induced asthma. To further explore the role of CD8+ T activation modified DCs in the asthma development, activated CD8+ T supernatant modified DCs (SN-OVA-DC) were transferred to induce asthma. The data in Fig. 6E showed lower levels of inflammatory cytokines IL-4 and IL-5 in BAL fluid in SN-OVA-DC group than control OVA-DC mice. These results indicated that the capability of SN-OVA-DC in priming OVA antigen specific CD4+ T cells proliferation was attenuated in vivo.

4. Discussion In this study, we reported that substantial IFN-c secreted by activated CD8+ T drove DCs into a tolerogenic form with up-regulated expression of iNOS, IDO and TGF-b, leading to CD4+ T cells proliferation inhibition. Further investigation had suggested that these activated CD8+ T therapeutically ameliorated CD4+ T-dependent allergic airway inflammation in an asthma model. Thus, we identified the immune regulatory functions of activated CD8+ T on CD4+ T cells proliferation, which through modifying the properties of DCs. The immune regulatory functions of CD8+ T have been widely explored in recent decades. Multiple regulatory CD8+ T subsets, including Qa-1 restricted regulatory CD8+ T [9], CD8+CD122+ T [31], CD8+CD103+ T [32], CD8+CD28 T [33], CD8+CD25+FoxP3+ T [34] and IL-10-secreting CD8+ T [35], had garnered much attention for their roles in maintenance of immune homeostasis. Different mechanisms also had been explored to explain the regulatory functions of these regulatory CD8+ T subsets, such as cell–cell contact, immunosuppressive factor induction or secretion, and even cytotoxicity dependence [1,8]. Here, different from those previous special regulatory CD8+ T subsets, we investigated the direct feedback regulation of activated CD8+ T population. Interestingly, these activated CD8+ T cells only exerted inhibitory functions on CD4+ T cells proliferation, but had little effect on its activation (Fig. 2A). Moreover, neither CD4+ Treg differentiation nor direct cytotoxicity on DCs mediated by CD8+ T was observed in vitro (Figs. 2B and 3), which was consistent with previous reports [36,37]. In fact, we

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

6

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

Fig. 3. The immune suppression of activated CD8+ T depended on soluble factors. (A) CD4+ T cells proliferation primed by polyformaldehyde-fixed maDC were suppressed by activated CD8+ T. CFSE-labeled OT-2 CD4+ T cells (2  105) were cultured in the presence or absence of OT-1 CD8+ T cells (1  105) with OVApep-loaded polyformaldehydefixed maDCs (2  104) in 96-well U-bottom plates for 96 h. Cell divisions of CD4+ T were analyzed with FACSAria. Data were shown as described in Fig. 1A. (B) The cytotoxicity of activated CD8+ T on maDCs. After being loaded with different doses of OVA257–264, 1  104 CMFDA-labeled maDCs were co-cultured with OT-1 CD8+ T at different CD8+ T/maDC ratios (5:1, 10:1, 20:1). After being cultured for 48 h, cells were collected and stained with PI for flow cytometer analysis. The absolute number of CMFDA+ PI living maDCs in each group was calculated. Data were presented as mean ± SEM (n = 3). (C) Phagocytosis of DCs. Different DCs co-cultured with OVA-Alexa488 and the fluorescence signals were detected using flow cytometer. (D) The regulation of activated CD8+ T on splenic CD11c+ DC subsets. After being co-cultured for 48 h, the expressions of CD86 and MHC on splenic CD11c+ DCs were detected using flow cytometer. (E) The immune suppression of activated CD8+ T cells was analyzed by transwell. In transwell group, CD4+ T/OVApep-loaded maDCs (1  106/1  105) in upper chamber of transwell, CD8+ T/OVApep-loaded maDCs (1  106/1  105) in lower well. After being cultured for 96 h, the divisions of CFSE+CD4+ T cells were analyzed by FCM. Data were shown as described in Fig. 1A. Results were from one representative of three performed experiments.

found that these activated CD8+ T exerted feedback regulation by modifying DCs into tolerogenic sets, which with less-efficient capability to prime CD4+ T cells into proliferation. As one heterogenerous population, DC subsets not only showed different roles in immunity priming or suppression, these cells also possessed highly plastic characteristics, and could modify its phenotype to get acclimatized to microenvironment. Unlike conventional DCs, which priming T cells activation and proliferation efficiently, tolerogenic DC subpopulations display low expression levels of MHC and co-stimulatory molecules, and have less capabilities in priming T cells proliferation [13,38]. Our previous studies had shown that tissue microenvironment could differentiate DCs

into DCreg, which immunoregulatory functions largely depended on cell–cell contact and soluble factors secretion [15,16]. Indeed, this study showed that immune microenvironment in activated CD8+ T derived supernatant also can modify the phenotype of conventional DCs into tolerogenic cells, which expressed low levels of I–Ab, CD80 and CD86 (Fig. 4C). Moreover, these SN-modified DCs up-regulated the expression levels of soluble immunoregulatory factors, including IDO, iNOS and TGF-b, which could efficiently suppressed CD4+ T responses [13,15,39]. Interestingly, the tolerogenic properties of SN-modified DCs could be partly abrogated by NO synthase inhibitor (PBIT) and IDO inhibitor (1-MT) respectively, but no similar effects were observed in TGF-b neutralizing

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

7

Fig. 4. Activated CD8+ T derived soluble factors modified maDCs phenotype. (A) The priming ability of maDCs on CD4+ T cells proliferation was attenuated by CD8+ T derived supernatant (SN) in vitro. OT-1 CD8+ T/maDC (1  106/1  105 per well) co-cultured for 36 or 60 h, and SN was collected for further experiments. SN-modified DCs were prepared by incubating OVA323–339-loaded maDCs with RPMI-1640 complete medium and activated CD8+ T derived SN mixture (1:1, v/v) for 48 h. After being cultured with DCs (unmodified or SN-modified) for 72 h, the divisions of CD4+ T were analyzed by flow cytometer. (B) SN-modified DCs showed lower capability than unmodified DCs in priming OVA323–339 CD4+ T cells into proliferation in vivo. Data in the plots were shown as mean ± SEM (n = 4), and the number in graph shown the percentage of CD4+KJ1-26+ T cells in total CD4+ T. (C) SN-modified DCs were prepared by incubating OVA-loaded DCs with RPMI-1640 complete medium and SN mixture (1:1, v/v) for 48 h. Cytokine production (IL-2 and IFN-c) of CD4+ T stimulated by SN-modified and unmodified DCs were analyzed using a mouse Th1/Th2/Th17/Th22 13plex flowcytomix assay kit. (D) SN-modified DCs showed less capability in priming OVA257–264 specific CD8+ T proliferation. Cell divisions of CD8+ T stimulated by SN-modified DCs were analyzed by detecting CFSE dye dilution with FACSAria after 72 h culture. (E) Phenotype of DCs. Before maDCs were prepared as below, these maDCs were firstly stained with Far-Red, and then were cultured under different treatment for 48 h. The phenotypes of these DCs were further analyzed by flow cytometer. CD8+ T-reacted DCs: OVA257–264-loaded maDCs co-cultured with OT-1 CD8+ T cells; SN-modified DCs: OVA257–264-loaded maDCs incubated with activated CD8+ T derived supernatant. Control maDCs: maDCs without any treatment. The numbers in graphs mean the fluorescence of B7-H1, CD11c, CD80, CD86 and I–Ab respectively. (F) The expressions of iNOS, IDO, TGF-b, IL-10, IL-12 and pSTAT3 in maDCs and SN-DCs were detected using Western blotting. (G) The cell divisions (left) and absolute cell numbers (right) of CD4+ T cells primed by DCs with/without different inhibitors. Numbers in histograms indicated the percentages of divided CD4+ T cells. Data were shown as mean ± SEM (n = 3). N.S., no significance; **p < 0.01. Data were one representative of at least three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

8

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

Fig. 5. IFN-c contributed to maDC modification. (A) The concentration of different cytokines in activated CD8+ T derived SN were analyzed. CD8+ T cells activated for 36 or 60 h, and the supernatants (36 or 60 h) were collected for cytokine profiles analysis. (B) Effects of exogenous IFN-c and IL-6 on OT-2 CD4+ T cells proliferation. Different doses of IL-6 and IFN-c were added to CD4+ T/maDC and the divisions of CD4+ T cells were analyzed by flow cytometer after being cultured for 72 h. Data were shown as mean ± SEM, and the numbers in graphs indicated the percentages of divided CD4+ T cells. (C and D) Effects of exogenous IFN-c on the CD4+ T cells proliferation which primed by fixed maDC (C) or IFN-cR(/) maDC (D). Absolute cell numbers counting (C) or the divisions (D) of CD4+ T were assessed by flow cytometer after being cultured for 72 h. (E) CD4+ T cells proliferation primed by SN modified DCs with/without IFN-c blocking. Results were from one representative of three performed experiments, and the data were shown as mean ± SEM (n = 3).

antibody. So, it suggested that IDO and NO contributed to the immune regulation of these tolerogenic DCs. STAT3 phosphorylation has been identified to be closely associated with nitric oxide secretion of DCs, and has the capability to control the release of TGF-b and IDO [26,27]. In addition, other studies also have demonstrated that STAT3 phosphorylation was involved in substantial down-regulated expression of co-stimulatory molecules and HLA-DR on DC membrane [40]. Indeed, these SN-modified-DCs expressed high level of phosphorylated signal transducers and activators of transcription 3 (pSTAT3) (Fig. 4F). Thus, to SN-modified DCs, the up-regulation of immunoregulatory factors, the down-regulation of surface co-stimulatory molecules and MHC class II were surmised to be associated with cytoplasmic pSTAT3 increase. Many factors were reported having contribution in the differentiation of tolerogenic DCs, including TGF-b, IL-10, HGF, M-CSF, VEGF, IL-6 and small metabolic molecules such as adenosine, progesterone and PGE2 [41–43]. Meanwhile, some studies also

showed that the immune regulation of IFN-c depended on DC modification with NO or IDO production [44,45]. Similarly, our study found that activated CD8+ T induced DCs with tolerogenicity through an IFN-c dependent manner in vitro. Interestingly, previous reports indicated that IFN-c could attenuate CD4+ T mediated airway inflammation by counteracting the Th2 response, remodeling invariant NKT cells, inhibiting eosinophil differentiation, and having direct effects on airway epithelium [46–49]. But, here we showed that CD8+ T activation in vivo, which resulted in a substantial secretion of IFN-c in BAL, could alleviate airway inflammation in OVA-induced asthma in CD8+ T deficient mice. These different mechanisms, which were reported to do contribution in the immunoregulation of IFN-c in inflammation of asthma, can be explained by the difference in its research models. Actually, some studies have reported that airway inflammation can be alleviated through DC modification, such as immunoregulatory gene transduction [50], and TGF-b pre-treatment [51]. Injection of plasmid with the gene encoding for IFN-c can alleviate airway allergic

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

9

Fig. 6. The regulatory function of activated CD8+ T on OVA-induced asthma. (A) Diagram of OVA-induced experimental asthma and OVA antigen specific CD8+ T interventional treatment. A total of 4  106 OVA specific OT-1 CD8+ T cells and 2  106 OVA257–264-loaded maDCs were infused i.v. 24 h prior to OVA inhalational challenge. Mice challenged with myelin oligodendrocyte glycoprotein (MOG) served as control mice. (B) The total cell number and composition of WBC in bronchoalveolar lavage (BAL) fluid of different mice. (C) The levels of IL-4, IL-5, IL-13 and IFN-c in BAL fluid were detected using a flowcytomix assay kit. (D) Histology of lung tissues. Lung tissue sections were stained by hematoxylin-eosin (H&E) (a–f) or periodic acid-Schiff (PAS) staining (g–l). (E) The levels of IL-4 and IL-5 in BAL fluid were detected after activated CD8+ T supernatant modified DCs being transferred. Magnification: 100 for H&E staining and 200 for PAS staining. Bar in the graphs: 50 lm. Large arrows: infiltrated leukocytes; small arrows: PAS+ goblet cells. i.p., intraperitoneally; i.v., intravenously; WBC, white blood cells; WT, wilde type; Eos, eosinophils; Lym, lymphocytes; Mon/Mac, monocytes/macrophages; Neu, neutrophils; OVA-DC, OVA loaded DC; SN-OVA-DC, activated CD8+ T supernatant treated OVA loaded DC, MOG-DC, MOG loaded DC; SN-MOGDC, activated CD8+ T supernatant treated MOG loaded DC, NS, no significance; data were shown as mean ± SEM (n = 5). *p < 0.05; **p < 0.01. All data were from one representative experiments, and repeated three times.

inflammation by suppressing the functions of CD11c+ APCs and their migration into bronchial lymph nodes [47]. In this study, we also surmised that the airway inflammation in asthma was

inhibited by activated CD8+ T derived IFN-c in a similar manner. Although the data showed IFN-c might have function in DCs modification and then suppressed antigen specific CD4+ T cells

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

10

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx

proliferation (Fig. 5), its underlying mechanisms still require further exploration. Moreover, CD8+ T infusion experiments on murine asthma model showed that CD8+ T activation can effectively inhibit the allergic CD4+ T responses (Fig. 6). We also found that activated CD8+ T supernatant-modified DCs had less effective ability in inducing asthma, with less IL-4 and IL-5 production in bronchial and lung in mice. However, which DC subset was exactly modified by activated CD8+ T in vivo, and then reduced allergy development still needs further exploration. Collectively, our results suggested that the soluble factors secreted by activated CD8+ T, including IFN-c, worked as immune regulator, which in turn modified mature DCs with tolerogenic properties, thereby suppressed the CD4+ T immune responses in vitro and in vivo. Therefore, DC modification by IFN-c derived from activated CD8+ T provides a promising immunotherapeutic target for the treatment of asthma and other CD4+ T mediated immune diseases. Conflict of interest The authors declare no conflict of interest for this work. Acknowledgments We thank Guangchao Li, Shaowei Wang, and Jie Feng for their kind and excellent technical assistance. We are also grateful to Chao Wang, Xi Liu, Wengang Song, Zhubo Chen, and Zhihai Qin for their helpful discussion and suggestion. We would like to thank Editage for providing editorial assistance. This study was kindly supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China (2012ZX09103301-017) and the General Program of National Natural Science Foundation of China (81272321, 81172834). References [1] G. Filaci, D. Fenoglio, F. Indiveri, CD8(+) T regulatory/suppressor cells and their relationships with autoreactivity and autoimmunity, Autoimmunity 44 (2011) 51–57, http://dx.doi.org/10.3109/08916931003782171. [2] K. Tsuchiya et al., Depletion of CD8+ T cells enhances airway remodelling in a rodent model of asthma, Immunology 126 (2009) 45–54, http://dx.doi.org/ 10.1111/j.1365-2567.2008.02876.x. [3] B.J. Skaggs, R.P. Singh, B.H. Hahn, Induction of immune tolerance by activation of CD8+ T suppressor/regulatory cells in lupus-prone mice, Hum. Immunol. 69 (2008) 790–796, http://dx.doi.org/10.1016/j.humimm.2008.08.284. [4] G. Das et al., Pivotal roles of CD8+ T cells restricted by MHC class I-like molecules in autoimmune diseases, J. Exp. Med. 203 (2006) 2603–2611, http:// dx.doi.org/10.1084/jem.20060936. [5] I. Menager-Marcq, C. Pomie, P. Romagnoli, J.P. van Meerwijk, CD8+CD28regulatory T lymphocytes prevent experimental inflammatory bowel disease in mice, Gastroenterology 131 (2006) 1775–1785, http://dx.doi.org/10.1053/ j.gastro.2006.09.008. [6] N. Najafian et al., Regulatory functions of CD8+CD28- T cells in an autoimmune disease model, J. Clin. Invest. 112 (2003) 1037–1048, http://dx.doi.org/ 10.1172/JCI17935. [7] N.R. York et al., Immune regulatory CNS-reactive CD8+T cells in experimental autoimmune encephalomyelitis, J. Autoimmun. 35 (2010) 33–44, http:// dx.doi.org/10.1016/j.jaut.2010.01.003. [8] C. Pomie, I. Menager-Marcq, J.P. van Meerwijk, Murine CD8+ regulatory T lymphocytes: the new era, Hum. Immunol. 69 (2008) 708–714, http:// dx.doi.org/10.1016/j.humimm.2008.08.288. [9] H.J. Kim, H. Cantor, Regulation of self-tolerance by Qa-1-restricted CD8(+) regulatory T cells, Semin. Immunol. 23 (2011) 446–452, http://dx.doi.org/ 10.1016/j.smim.2011.06.001. [10] A. Noble, A. Giorgini, J.A. Leggat, Cytokine-induced IL-10-secreting CD8 T cells represent a phenotypically distinct suppressor T-cell lineage, Blood 107 (2006) 4475–4483, http://dx.doi.org/10.1182/blood-2005-10-3994. [11] S. Laffont et al., CD8+ T-cell-mediated killing of donor dendritic cells prevents alloreactive T helper type-2 responses in vivo, Blood 108 (2006) 2257–2264, http://dx.doi.org/10.1182/blood-2005-10-4059. [12] D. Kagi et al., Fas and perforin pathways as major mechanisms of T cellmediated cytotoxicity, Science 265 (1994) 528–530. [13] A.E. Morelli, A.W. Thomson, Tolerogenic dendritic cells and the quest for transplant tolerance, Nat. Rev. Immunol. 7 (2007) 610–621, http://dx.doi.org/ 10.1038/nri2132.

[14] F. Blengio et al., The hypoxic environment reprograms the cytokine/chemokine expression profile of human mature dendritic cells, Immunobiology 218 (2013) 76–89, http://dx.doi.org/10.1016/j.imbio.2012.02.002. [15] M. Zhang et al., Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells, Nat. Immunol. 5 (2004) 1124–1133, http:// dx.doi.org/10.1038/ni1130. [16] Q. Li, Z. Guo, X. Xu, S. Xia, X. Cao, Pulmonary stromal cells induce the generation of regulatory DC attenuating T-cell-mediated lung inflammation, Eur. J. Immunol. 38 (2008) 2751–2761, http://dx.doi.org/10.1002/ eji.200838542. [17] K. Zhong et al., Murine myeloid dendritic cells that phagocytose apoptotic T cells inhibit the immune response via NO, PLoS One 7 (2012) e49378, http:// dx.doi.org/10.1371/journal.pone.0049378. [18] M. Svensson, A. Maroof, M. Ato, P.M. Kaye, Stromal cells direct local differentiation of regulatory dendritic cells, Immunity 21 (2004) 805–816, http://dx.doi.org/10.1016/j.immuni.2004.10.012. [19] S.R. Clarke et al., Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection, Immunol. Cell Biol. 78 (2000) 110–117, http://dx.doi.org/10.1046/j.1440-1711.2000.00889.x. [20] J.M. Robertson, P.E. Jensen, B.D. Evavold, DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323–339 epitope, J. Immunol. 164 (2000) 4706–4712. [21] B.H. Koller, P. Marrack, J.W. Kappler, O. Smithies, Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells, Science 248 (1990) 1227–1230. [22] S. Huang et al., Immune response in mice that lack the interferon-gamma receptor, Science 259 (1993) 1742–1745. [23] E.M. Shevach et al., The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells, Immunol. Rev. 212 (2006) 60–73, http://dx.doi.org/10.1111/ j.0105-2896.2006.00415.x. [24] F.F. de Araujo et al., Foxp3+CD25(high) CD4+ regulatory T cells from indeterminate patients with Chagas disease can suppress the effector cells and cytokines and reveal altered correlations with disease severity, Immunobiology 217 (2012) 768–777, http://dx.doi.org/10.1016/ j.imbio.2012.04.008. [25] H. Dong, X. Chen, Immunoregulatory role of B7–H1 in chronicity of inflammatory responses, Cell. Mol. Immunol. 3 (2006) 179–187. [26] P. Bonifazi et al., Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans, Mucosal Immunol. 2 (2009) 362–374, http://dx.doi.org/10.1038/mi.2009.17. [27] H. Shimizu, M. Yisireyili, F. Nishijima, T. Niwa, Stat3 contributes to indoxyl sulfate-induced inflammatory and fibrotic gene expression and cellular senescence, Am. J. Nephrol. 36 (2012) 184–189, http://dx.doi.org/10.1159/ 000341515. [28] J.Y. Niederkorn, Emerging concepts in CD8(+) T regulatory cells, Curr. Opin. Immunol. 20 (2008) 327–331, http://dx.doi.org/10.1016/j.coi.2008.02.003. [29] J.L. Ingram, M. Kraft, IL-13 in asthma and allergic disease: asthma phenotypes and targeted therapies, J. Allergy Clin. Immunol. 130 (2012) 829–842, http:// dx.doi.org/10.1016/j.jaci.2012.06.034 (quiz 843-824). [30] F.D. Finkelman, S.P. Hogan, G.K. Hershey, M.E. Rothenberg, M. Wills-Karp, Importance of cytokines in murine allergic airway disease and human asthma, J. Immunol. 184 (2010) 1663–1674, http://dx.doi.org/10.4049/ jimmunol.0902185. [31] A.T. Endharti et al., CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis, J. Immunol. 186 (2011) 41–52, http://dx.doi.org/10.4049/jimmunol.1000800. [32] M. Mikko et al., Increased intraepithelial (CD103+) CD8+ T cells in the airways of smokers with and without chronic obstructive pulmonary disease, Immunobiology 218 (2013) 225–231, http://dx.doi.org/10.1016/ j.imbio.2012.04.012. [33] M. Strioga, V. Pasukoniene, D. Characiejus, CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease, Immunology 134 (2011) 17–32, http://dx.doi.org/10.1111/j.1365-2567.2011.03470.x. [34] L. Cosmi et al., Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes, Blood 102 (2003) 4107–4114, http://dx.doi.org/10.1182/blood-2003-04-1320. [35] M. Abel et al., Intrahepatic virus-specific IL-10-producing CD8 T cells prevent liver damage during chronic hepatitis C virus infection, Hepatology 44 (2006) 1607–1616, http://dx.doi.org/10.1002/hep.21438. [36] R.A. Willis, J.W. Kappler, P.C. Marrack, CD8 T cell competition for dendritic cells in vivo is an early event in activation, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 12063–12068, http://dx.doi.org/10.1073/pnas.0605130103. [37] I. Nordstrom, M. Nurkkala, L.V. Collins, K. Eriksson, CD8+ T-cells suppress antigen-specific and allogeneic CD4+ T-cell responses to herpes simplex virus type 2-infected human dendritic cells, Viral Immunol. 18 (2005) 616–626, http://dx.doi.org/10.1089/vim.2005.18.616. [38] L.M. van Duivenvoorde, G.J. van Mierlo, Z.F. Boonman, R.E. Toes, Dendritic cells: vehicles for tolerance induction and prevention of autoimmune diseases, Immunobiology 211 (2006) 627–632, http://dx.doi.org/10.1016/ j.imbio.2006.05.014. [39] A.L. Mellor, D.H. Munn, IDO expression by dendritic cells: tolerance and tryptophan catabolism, Nat. Rev. Immunol. 4 (2004) 762–774, http:// dx.doi.org/10.1038/nri1457. [40] J. Correale, A. Villa, Role of CD8+ CD25+ Foxp3+ regulatory T cells in multiple sclerosis, Ann. Neurol. 67 (2010) 625–638, http://dx.doi.org/10.1002/ ana.21944.

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001

D. Chen et al. / Cellular Immunology xxx (2015) xxx–xxx [41] S. Xia et al., Hepatic microenvironment programs hematopoietic progenitor differentiation into regulatory dendritic cells, maintaining liver tolerance, Blood 112 (2008) 3175–3185, http://dx.doi.org/10.1182/blood-2008-05159921. [42] K. Okunishi et al., A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function, J. Immunol. 175 (2005) 4745–4753. [43] C. Menetrier-Caux et al., Inhibition of the differentiation of dendritic cells from CD34(+) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor, Blood 92 (1998) 4778–4791. [44] A.J. Sant, F.A. Chaves, S.A. Leddon, J. Tung, The control of the specificity of CD4 T cell responses: thresholds, breakpoints, and ceilings, Front. Immunol. 4 (2013) 340, http://dx.doi.org/10.3389/fimmu.2013.00340. [45] G. Ren et al., Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide, J. Immunol. 181 (2008) 3277–3284. [46] A. Dubois et al., Regulation of Th2 responses and allergic inflammation through bystander activation of CD8+ T lymphocytes in early life, J. Immunol. 185 (2010) 884–891, http://dx.doi.org/10.4049/jimmunol.0903287.

11

[47] A.M. de Bruin et al., Eosinophil differentiation in the bone marrow is inhibited by T cell-derived IFN-gamma, Blood 116 (2010) 2559–2569, http://dx.doi.org/ 10.1182/blood-2009-12-261339. [48] H. Matsuda et al., Plasticity of invariant NKT cell regulation of allergic airway disease is dependent on IFN-gamma production, J. Immunol. 185 (2010) 253– 262, http://dx.doi.org/10.4049/jimmunol.0902301. [49] C. Mitchell, K. Provost, N. Niu, R. Homer, L. Cohn, IFN-gamma acts on the airway epithelium to inhibit local and systemic pathology in allergic airway disease, J. Immunol. 187 (2011) 3815–3820, http://dx.doi.org/10.4049/ jimmunol.1100436. [50] E. Henry et al., Dendritic cells genetically engineered to express IL-10 induce long-lasting antigen-specific tolerance in experimental asthma, J. Immunol. 181 (2008) 7230–7242. [51] J. Zhang-Hoover, P. Finn, J. Stein-Streilein, Modulation of ovalbumin-induced airway inflammation and hyperreactivity by tolerogenic APC, J. Immunol. 175 (2005) 7117–7124.

Please cite this article in press as: D. Chen et al., CD8+ T activation attenuates CD4+ T proliferation through dendritic cells modification, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.05.001