Introduction
Rheumatoid arthritis (RA) is characterised by progressive joint inflammation that results in tissue damage [
1]. This is strongly dependent on CD4 T cell production of Th1 (interferon γ) and Th17 cytokines (interleukin 17 (IL-17)) [
2]-[
5]. Activation and differentiation of CD4 T cells to become Th1 or Th17 cells is strongly regulated by antigen-presenting cells such as dendritic cells (DCs) [
6]. Several types of DCs are known to circulate in human blood. They are characterised by high expression of human leucocyte antigen (HLA) class II molecules and the absence of lineage markers (CD3, CD19, CD14, CD20, CD56 and glycophorin A). Human blood DCs can be divided into at least three subtypes (plasmacytoid DCs and two types of myeloid or classical DCs (mDC1 and mDC2)) [
7],[
8], based on the blood-derived DC antigen (BDCA) molecules [
9],[
10]. BDCA-1 (CD1c) identifies the mDC1 subset, which comprises potent activators of CD4 T cells, whereas mDC2 cells, identified by expression of BDCA-3 (CD141), more potently activate CD8 T cells [
7],[
9],[
10]. In this respect, it is important to note that the characterisation of mDC1 cells by CD1c is more specific than the previously used and more broadly expressed marker, CD11c [
7],[
9]. CD1c mDCs are abundantly present in joints of RA patients, and these synovial fluid (SF)–derived mDCs have recently been demonstrated to have an extremely strong capacity to activate autologous peripheral blood (PB)–derived CD4 T cells [
11].
Thymic stromal lymphopoietin (TSLP) has recently been considered as a potential trigger to activate CD1c mDCs in the joints of RA patients. TSLP cytokine levels are significantly increased in the SF of RA patients compared with SF of osteoarthritis patients [
12],[
13]. TSLP has been demonstrated to potently activate TSLPR-expressing CD1c mDCs from SF to secrete enhanced levels of T cell–attracting chemokines and to strongly activate PB-derived CD4 T cells to induce Th1, Th17 and Th2 activity [
13]. In addition, recently, TSLP and its receptor were also shown to enhance Th1- and Th17-mediated experimental arthritis and tissue destruction [
14].
Because of the prominent role of CD4 T cells in arthritic processes and the potential of SF-derived mDCs and TSLP-primed mDCs to activate autologous PB-derived CD4 T cells, in this study we investigated the potential of these mDCs to activate autologous SF-derived CD4 T cells. An evident hyporesponsiveness of SF-derived CD4 T cells upon mDC or TSLP-primed mDC activation was observed. Several observations led us to investigate the role of programmed death 1 (PD-1) and its ligand interactions in this hyporesponsiveness, because ligation of PD-1 by PD-L1 or PD-L2 leads to inhibition of T cell proliferation [
15],[
16]. First, our analysis of the gene expression profiles of TSLP-primed mDCs from RA patients revealed significant upregulation of PD-L1 and much higher expression levels compared with PD-L2. In addition, preliminary data had shown us that PD-L1 was upregulated on SF mDCs of RA patients. Third, data from previous studies [
17] and ours indicated overexpression of PD-1 on synovial CD4 T cells of RA patients. Because IL-7 recently was shown to downregulate PD-1 expression on T cells [
18] and because of the potent T cell stimulatory capacity of IL-7 [
19], we also examined the role of IL-7 in the regulation of PD-1/PD-L interactions in hyporesponsiveness of synovial T cells. Our data demonstrate that hyporesponsiveness of synovial CD4 T cells in response to SF-derived CD1c mDCs is dependent on PD-1/PD-L1 interactions and is robustly reversed by IL-7.
Methods
Patients
SF was obtained from 13 RA patients during effusion of the knee and 70 ml of heparinized PB was collected from 16 RA patients. RA was classified according to the American College of Rheumatology criteria [
20]. Ethical approval for this study was granted by the medical ethics committee of the University Medical Center Utrecht for the collection of patient samples in compliance with the Helsinki Declaration. All patients gave their written informed consent to participate.
Cell isolation
Mononuclear cells (MNCs) were isolated from PB and SF by density centrifugation using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden). Prior to MNC isolation, PB or SF was diluted 1:1 with RPMI 1640 medium (Gibco/Life Technologies, Grand Island, NY, USA) containing penicillin (100 U/ml), streptomycin (100 μg/ml) and glutamine (2 mM) (all from PAA Laboratories, Pasching, Austria). PB- and SF-derived CD1c mDCs and CD4 T cells were isolated from the MNC fraction by magnetic-activated cell sorting (MACS) using CD1c (BDCA-1)-positive dendritic cell and CD4+ T cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany). PB memory T cells were isolated from the CD4 T cell fraction by their lack of CD45RA expression using the CD45RA+ isolation kit (Miltenyi Biotec). Isolations were performed according to the manufacturer’s instructions.
Cell cultures
All cells were cultured in RPMI GlutaMAX medium (Gibco/Life Technologies) supplemented with penicillin, streptomycin and 10% (vol/vol) GemCell human serum AB (Gemini Bio-Products, West Sacramento, CA, USA). The mDCs were cultured at a cell concentration of 0.5 × 106 cells/ml with or without recombinant TSLP at 20 ng/ml (R&D Systems, Minneapolis, MN, USA) for 20 hours in Sarstedt tubes (Sarstedt, Nümbrecht, Germany) at 37°C.
The functional capacities of PB- and SF-derived mDCs were assessed by measuring activation of autologous CD4 T cells derived from PB and SF. For this purpose, isolated T cells were seeded in round-bottomed 96-well plates at a concentration of 0.25 × 106 cells/ml and kept at 37°C in full culture medium before coculturing with the (TSLP-primed) mDCs. Washed TSLP-activated mDCs and unstimulated mDCs were added to the autologous T cells (mDC:T cell ratio 1:5 or 1:10) in triplicate in the absence of additional stimuli and cocultured for 6 days. To test the effects of PD-1 blockade or IL-7 effects in cocultures of mDCs and memory T cells from PB and SF, anti-PD-1 mAB (1 μg/ml; BioLegend, San Diego, CA, USA) or IL-7 (10 ng/ml, PeproTech, Rocky Hill, NJ, USA) was added at the start of the cocultures. Proliferation was measured by [3H]thymidine incorporation (1 μCi/well added during the last 18 hours of a culture period; PerkinElmer, Waltham, MA, USA).
Flow cytometry
PD-1 expression on CD4 T cells and PD-L1 expression on CD1c mDCs of RA patients were analysed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Ex vivo or cultured mDCs were stained with CD1c phycoerythrin (CD1c-PE; BD Biosciences), CD19 peridinin chlorophyll (CD19-PerCP; BioLegend) and CD274 (PD-L1)-APC (BioLegend). mDCs were gated as CD1c-positive and CD19-negative. Ex vivo CD4 T cells were stained with CD45RO fluorescein isothiocyanate (CD45RO-FITC; Dako, Glostrup, Denmark), CD27-APC (Invitrogen), CD279 (PD-1)-PE and CD4-PerCP (BioLegend) using isotype antibodies or autofluorescence as controls. All samples were analysed using FlowJo software (TreeStar, Ashland, OR, USA). To compare mean fluorescence intensity (MFI) values, the autofluorescence intensity was subtracted from the MFI of the stains to reveal true expression values.
Real-time quantitative RT-PCR
To examine TSLP-mediated PD-L1 gene regulation in isolated CD1c mDCs from PB and SF of RA patients, CD1c mDCs (0.5 × 10
6 cells/ml) were stimulated with 20 ng/ml recombinant TSLP (R&D Systems) or left untreated for 20 hours in Sarstedt tubes at 37°C. mDCs were collected and lysed in Buffer RLT (QIAGEN, Carpinteria, CA, USA) prior to RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. RNA quantity was measured by using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and RNA quality was assessed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA). cDNA synthesis was performed with an automated system (Caliper Life Sciences NV/SA, Teralfene, Belgium), starting with 70 ng of total RNA from each sample, as previously described in detail [
21]. Gene expression was quantified by quantitative RT-PCR (RT-qPCR) performed in duplicate from 3 ng of cDNA in the presence of SYBR Select Master Mix (Life Technologies, Carlsbad, CA, USA) and 1.5 μM of specific primer pairs (PD-L1 forward: 5′-GCT GAA CGC ATT TAC TGT CAC-3′, PD-L1 reverse: 5′-TGT TCT TAT CCT CCA TTT CCC A-3′; GAPDH forward: 5′-ATG GGG AAG GTG AAG GTC G-3′, GAPDH reverse: 5′-GGG GTC ATT GAT GGC AAC AAT A-3′). The analyses were performed using the QuantStudio 12K Flex system (Life Technologies) with the following thermal cycle conditions: 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. Gene expression values were calculated according to the comparative threshold cycle method [
22] using the stably expressed GAPDH as an endogenous control. The value of each control sample was set at 1 and was used to calculate the fold change in target mRNA.
Statistical analysis
Differences between conditions were assessed by paired-samples evaluation using the nonparametric Wilcoxon signed-rank test unless indicated otherwise. Unpaired data analysis was performed using the nonparametric Mann–Whitney U test. Data analysis was performed using SPSS software version 20.0 (SPSS, Chicago, IL, USA). Data were considered statistically significant at P < 0.05.
Discussion
In the present study, we have demonstrated that, in contrast to CD4 T cells from PB, CD4 T cells derived from SF are hyporesponsive upon activation by (TSLP-primed) CD1c mDCs. SF-derived CD4 T cells consist mainly of memory T cells, which express elevated levels of PD-1, whereas SF-derived CD1c mDCs abundantly express its ligand PD-L1, which is upregulated by TSLP. Memory T cell hyporesponsiveness is partially dependent on PD-1/PD-L1 interactions because blockade of these interactions by anti-PD-1 antibodies partially restores the T cell proliferation. Importantly, IL-7, an inhibitor of PD-1 expression, also overcomes hyporesponsiveness and robustly activates synovial memory T cells in the context of activated (TSLP-primed) mDCs, which could be further enhanced by blocking PD-1.
Similarly to TSLP-primed mDCs from PB, SF-derived mDCs have a strongly enhanced capacity to activate T cells derived from PB that is even upregulated upon TSLP stimulation [
13]. Associated with this enhanced T cell stimulatory capacity, enhanced expression of antigen-presenting molecules, including CD1c and HLA class II, and costimulatory molecules, including CD80, CD86 and CD40, have been demonstrated [
11],[
13]. In support of this finding, activation of PB-derived T cells by (TSLP-primed) mDCs is strongly dependent on costimulation and antigen presentation as blockade of HLA-class II molecules or CD80/86 costimulation blocked T cell activation [
13]. However, despite the abundance of antigen-presenting and costimulatory molecules, the activity of T cells derived from SF of RA patients activated by autologous (TSLP-primed) mDCs is strongly hampered. Although several mechanisms might be involved in the reduced capacity of SF-derived T cells in response to activated mDCs, the role of PD-1/PD-L1 interactions, which negatively regulate T cell activation [
15], was investigated for obvious reasons, such as overexpression of PD-L1 by activated mDCs from SF and high PD-L1 mRNA levels induced by TSLP. Blockade of PD-1/PD-L1 interactions has been shown to result in increased T cell activity [
23] and PD-1 deficiency, and PD-L1 deficiency is shown to play a critical role in the development of autoimmune disorders in mice [
24],[
25].
In line with our results showing increased PD-1 expression on SF-derived memory T cells compared with PB-derived T cells, PD-1 expression was previously described to be increased on SF-derived CD4 T cells [
17]. These SF-derived T cells were previously suggested to be resistant to PD-1 mediated suppression, as PD-L1 expression on SF-derived CD14+ monocytes and macrophages was not sufficiently high to effectively downregulate T cell activation [
17]. In contrast to this latter suggestion, we observed hyporesponsiveness of SF T cells upon activation by
in vivo activated synovial mDCs and
in vitro activated TSLP-mDCs, which, in contrast, expressed high levels of PD-L1. PD-1-blocking antibodies, blocking interactions with PD-L1, resulted in an increase in the T cell proliferation, indicating that SF-derived T cell hyporesponsiveness is at least partially dependent on PD-1/PD-L interactions. However, based on the strong expression of both PD-1 on T cells and PD-L1 on activated mDCs, more robust reversal of T cell hyporesponsiveness was expected. The lack of response might be due to the fact that PD-1 is also expressed on regulatory T cells, preventing immunosuppression by these cells [
26]. Because previous studies, including those of our group, have demonstrated an increased presence of regulatory T cells in the SF [
27], blockade of PD-1, next to increased activation of effector cells, might enhance the function of regulatory T cells, resulting in a somewhat more limited reversal of T cell inhibition. An alternative explanation is that enhanced antigen presentation due to robust upregulation of HLA class II by TSLP may limit the effect of PD-1 blockade, which is consistent with previous data demonstrating that the inhibitory effect of the PD-1/PD-L pathway on CD4 T cells was greatest at lower antigen concentrations [
16],[
25]. Additionally, blockade of PD-L1 has been shown to more potently restore T cell proliferation compared with PD-1 blockade in antigen-driven T cell responses [
28]. Although not tested in the present study, this might result in a stronger restoration of the SF-derived T cell hyporesponsiveness.
Hyporesponsiveness of SF-derived CD4 T cells upon mDC or TSLP-mDC stimulation was robustly reversed by IL-7, which is in line with previous data demonstrating vigorous activation and even hyperresponsiveness of synovial CD4 T cells in the context of monocytic cells [
19]. Recently, IL-7 was shown to prevent shutdown of T cell activation by downregulation of PD-1 expression [
18]. We also found PD-1 downregulation upon IL-7 stimulation of CD4 T cells in the present study (data not shown). Apart from the capacity of IL-7 to downregulate PD-1 expression [
18], IL-7 is also shown to overcome PD-1-mediated inhibition of T cells by STAT5 activation that is regulated by IL-2 and family members such as IL-7 and IL-15 [
29],[
30]. IL-7 and IL-15 are both abundantly present in joints of RA patients [
19],[
31], which suggests that the inflammatory environment in RA joints could play a critical role in overcoming PD-1-mediated inhibition of T cells. Although reversing PD-1-mediated inhibition is likely a crucial mechanism whereby IL-7 overcomes hyporesponsiveness, other mechanisms induced by IL-7 could be involved as well, such as upregulation of (co)stimulatory molecules that were previously found to be induced by IL-7, both on T cells (for example, lymphocyte function–associated antigen and CD69) and on myeloid cells (CD40, CD80, CD86) [
19].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FMM participated in the study design, acquisition of data, analysis and interpretation of data, and manuscript drafting. KMGWJ contributed to the acquisition of data and critical revision of the manuscript. JWJB and FPJGL contributed to the study design, interpretation of data and critically revision of the manuscript. JAGR contributed to the study design, analysis and interpretation of data, and manuscript drafting. All authors read and approved the final manuscript.