Alternatively activated dendritic cells derived from systemic lupus erythematosus patients have tolerogenic phenotype and function

Clinical Immunology (2014) 156, 43–57

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

Alternatively activated dendritic cells derived from systemic lupus erythematosus patients have tolerogenic phenotype and function Hai Jing Wu a , Yi Lo a , Daniel Luk a , Chak Sing Lau a , Liwei Lu b , Mo Yin Mok a,⁎ a b

Division of Rheumatology & Clinical Immunology, Department of Medicine, University of Hong Kong, Hong Kong Department of Pathology, University of Hong Kong, Hong Kong

Received 3 May 2014; accepted with revision 28 October 2014 KEYWORDS Dendritic cells; Regulatory T cell; Tolerogenicity; Vitamin D3;

Abstract Tolerogenic dendritic cells (DCs) are potential cell-based therapy in autoimmune diseases. In this study, we generated alternatively activated DCs (aaDCs) by treating monocyte-derived DCs from patients with systemic lupus erythematosus (SLE) and healthy subjects with combination of 1,25 dihydroxyvitamin D(3) (vitD3) and dexamethasone followed by lipopolysaccharide-induced maturation. Lupus aaDCs were found to acquire semi-mature phenotype that remained maturation-resistant to immunostimulants. They produced low level of IL-12 but high level of IL-10. They had attenuated allostimulatory effects on T cell activation and proliferation comparable to normal aaDCs and demonstrated differential immunomodulatory effects on naïve and memory T cells. These aaDCs were capable of inducing IL-10 producing regulatory T effectors from naïve T cells whereas they modulated cytokine profile with suppressed production of IFN-γ and IL-17 by co-cultured memory T cells with attenuated proliferation. These aaDCs were shown to be superior to those generated using vitD3 alone in lupus patients. © 2014 Elsevier Inc. All rights reserved.

Abbreviations: aaDCs, alternatively activated dendritic cells; matDCs, mature dendritic cells; Taa, T cells primed by alternatively activated dendritic cells; Tmat, T cells primed by mature dendritic cells; Tvd, T cells primed by vitD3-treated dendritic cells; TolDCs, tolerogenic dendritic cells; vdDCs, vitD3-treated dendritic cells ⁎ Corresponding author at: Division of Rheumatology & Clinical Immunology, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong. Fax: + 852 2872 5828. E-mail addresses: [email protected] (H.J. Wu), [email protected] (Y. Lo), [email protected] (D. Luk), [email protected] (C.S. Lau), [email protected] (L. Lu), [email protected] (M.Y. Mok).

http://dx.doi.org/10.1016/j.clim.2014.10.011 1521-6616/© 2014 Elsevier Inc. All rights reserved.

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1. Introduction Tolerogenic dendritic cells (TolDCs) have increasingly been studied as cell-based therapeutic regimen in murine models of autoimmune diseases such as diabetes [1], experimental autoimmune encephalitis [2] and inflammatory arthritis [3] with promising results. TolDCs can induce and maintain peripheral T cell tolerance by various mechanisms including induction of T cell deletion, anergy, cytokine deviation, and induction of regulatory T cells (Treg) [4]. TolDCs can be induced by cytokines such as interleukin (IL)-10 [5], tumour necrosis factor (TNF)-α [6] and pharmacological agents [7]. They are characterised by semi-mature phenotype with high expression of co-stimulatory molecules and MHC class II and production of low level pro-inflammatory cytokines such as IL-12, IL-6 and TNF-α [8]. Treatment with active metabolite of Vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH2)D3] (vitD3), and dexamethasone followed by lipopolysaccharide (LPS) maturation has previously been adopted to generate alternatively activated DCs (aaDCs) that are maturation-resistant with tolerogenic function [9,10]. Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by dysregulated innate and adaptive immune responses with immune-complexes formation leading to tissue inflammation and organ failure. Current treatment strategies for SLE involve high dose corticosteroid and immunosuppressants that are associated with significant adverse effects. Thus, induction of immune tolerance by tolDCs appears to be an appealing therapeutic tool for this disease. One issue unresolved in SLE patients in this regard was the altered phenotype of peripheral monocytes with accelerated differentiation and maturation into DCs with T cell activating capability [11–13]. It remains controversial if this over-stimulated phenotype of lupus monocyte arises as a result of inherent cellular abnormalities or secondary to an aberrant cytokine and chemokine environment [14]. In this study, we generated aaDCs by vitD3 and dexamethasone from SLE patients with quiescent disease and examined their phenotype and function as tolDCs. We found that aaDCs derived from lupus patients and normal subjects have comparable suppressive effects on allogeneic T cells, can polarise naïve T cells into IL-10 producing Treg and attenuate pro-inflammatory phenotype of memory T cells.

2. Material and methods 2.1. Patients and controls The study was approved by the ethics committee of the Institutional Review Board of The University of Hong Kong/ Hong Kong West Cluster with informed consent from recruited subjects. Patients who satisfied the American College of Rheumatology revised classification criteria for SLE [15] were recruited from the University affiliated lupus clinic. Disease activity was determined according to SLE disease activity index (SLEDAI) [16]. Monocytes and T cells were isolated from patients with quiescent disease for DC derivation and functional DC-T co-culture experiments. Sera was obtained from patients with inactive and active disease (SLEDAI N 6) for

H.J. Wu et al. DC challenge experiments. Age- and sex-matched healthy controls were recruited from staff clinic.

2.2. Generation of mature DCs, vitD3-treated DCs and alternatively activated DCs Peripheral blood mononuclear cells were isolated from fresh venous blood by density centrifugation on Ficoll-Paque (GE Healthcare, Sweden). CD14+ monocytes were isolated using magnetic microbeads (Miltenyi Biotec, Germany). Monocytederived DCs (MDDCs) were generated by culture of monocytes at 1 × 106 cells/ml in the presence of IL-4 and GM-CSF (20 ng/ml each, PeproTech, USA) for 9 days with refreshed medium and cytokines on day 5. Mature DCs (matDCs) were generated on day 8 by addition of LPS (50 ng/ml, Sigma, USA) for 24 h. To generate vitD3-treated DCs (vdDCs), 1,25(OH2)D3 (1 × 10−10 M, Sigma) was added on day 8 for 24 h in the presence of LPS (50 ng/ml). Alternatively activated DCs (aaDCs) were generated by treating DCs with dexamethasone (1 × 10−6 M, Sigma) on day 5, followed by dexamethasone (1 × 10−6 M), vitD3 (1 × 10−10 M) and LPS (50 ng/ml) on day 8 for 24 h. Cells were cultured in RPMI 1640 supplemented with 10% foetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2. In some experiments, MDDCs were challenged by soluble CD40L (sCD40L, 3 μg/ml, PeproTech), CpG-B DNA (10 μg/ml, Hycult Biotech, USA), R848 (1 μg/ml, InvivoGen, USA), interferon (IFN)-α (1000 U/ml, Millipore, USA), 25% inactive or active SLE serum.

2.3. DC-T co-culture experiments CD45RA+/RO− naïve and CD45RA−/RO+ memory CD4+ T cells were isolated by negative isolation kits (Miltenyi Biotec). MDDCs were co-cultured with allogeneic naïve or memory T cells (1:10) for 6 days. T cell proliferation was assessed by incorporation of 3H-thymidine for the last 18 h of culture measured by scintillation counting (Microbeta TriLux, USA). Recombinant human IL-12 (rIL-12) (5 ng/ml PeproTech), neutralizing anti-IL-10 (10 μg/ml) and isotypic antibody (R&D, USA) were used in some experiments.

2.4. T cell suppression assay CD4+CD25− responder T cells were isolated by depleting CD25hi cells using anti-CD25 beads (Miltenyi Biotec). Allogeneic naïve or memory T cells were primed by DCs (10:1) for 6 days and rested for 4 days in 50 ng/ml rIL-2 (PeproTech). Irradiated (50 Gy) MDDC-primed T cells were co-cultured with allogeneic CD4+CD25− responder T cells (1:2) for 5 days in the presence of matDCs (DC:responder T cells 1:10, 1:40, 1:80). CD4+CD25hi Treg enriched by Treg isolation kit (Miltenyi Biotec) were used as positive control.

2.5. Flow cytometry To examine the expression of surface markers and intracellular molecules, DCs or T cells were incubated with FcR blocking reagent (Miltenyi Biotec) for 10 min followed by primary antibodies on ice for 30 min. Antibodies used for

aaDCs derived from SLE patients have tolerogenic phenotype and function flow cytometry included HLA-DR-FITC (Beckman Coulter, USA), CD80-PE-Cy5, CD86-PE, CD40-PE-Cy5, CD83-PE, CD25-PE, CD69-PE-Cy5, CD4-FITC, CD4-Cy5 and isotypic controls (BD Biosciences, USA). Intracellular cytokine profile was studied using monoclonal antibodies including IFN-γ-APC, IL-4-PE, IL-10-Alexa Flour488 (BD Biosciences) and IL-17-PE (R&D). Cells were permeabilised and fixed by cytofix/cytoperm buffers (eBioscience, USA) with Golgistop added for the last 4 h. Apoptosis was detected by Annexin V Apoptosis Detection Kit II (BD Biosciences). Data was acquired by flow cytometry (Beckman coulter, FC500, USA) and analysed using FlowJo (Tree star, USA).

2.6. ELISA Supernatants were harvested from MDDC culture and stored at − 80 °C for subsequent cytokine measurement. Cytokines including IL-12p70, IL-6 and IL-10 were measured by sandwich ELISA (eBioscience).

2.7. Western blotting Protein in MDDC cell lysate was quantified by Bradford assay (HyClone-Pierce, USA) followed by 12% vertical dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, USA). The membrane was blocked in TBS/5% skim milk and then incubated with mouse anti-human RelB monoclonal antibody (Santa Cruz, USA) for 1 h followed by HRP-rabbit anti-mouse IgG antibody (Santa Cruz). Proteins were detected with ECL Western blot detection kit (Thermo Scientific, USA). Quantification of RelB was normalized to β-actin by densitometry.

2.8. Real-time PCR Total RNAs were extracted from MDDCs and MDDC-primed naïve T cells using Trizol kit (Sigma). First-strand cDNAs were synthesized using SuperScript® III Reverse Transcriptase kit (Invitrogen, USA) at 25 °C for 10 min, 50 °C for 50 min, and 85 °C for 5 min. Quantitative real-time PCR for RelB and IDO expression in DCs was performed using SYBR Green I Mastermix (Invitrogen) with forward and reverse primers (QuantiTect® primer Assay, Qiagen, USA). The PCR products were denatured for 10 min at 95 °C and then amplified over 40 cycles of 95 °C for 20 s and 60 °C for 30 s. Expression of T-bet, GATA-3, Foxp3 and RORγt in T cells was measured using Taqman Gene Expression Assay (Applied Biosystem, USA) with forward and reverse primers. The PCR products were denatured for 10 min at 95 °C and then amplified over 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Real-time PCR for all samples was run on DNA Engine Opticon 2 and values were calculated using 2−△Ct formula and normalized to GAPDH (Taqman® Pre-Developed Assay, Applied Biosystem).

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non-normally distributed data. P-value b 0.05 is considered statistically significant.

3. Results 3.1. Lupus aaDCs had semi-mature phenotype Expression of co-stimulatory molecules and maturation markers (CD80, CD86, CD40, CD83) in aaDCs, vdDCs and matDCs from healthy subjects and SLE patients was studied by flow cytometry to examine their maturity status. All expressed high levels of MHC Class II molecule (HLA-DR) (Fig. 1A). As expected, matDCs expressed high levels of maturation marker CD83 and co-stimulatory molecules that facilitate T cell activation and differentiation. Significantly lower CD83 were found in normal vdDCs by 2.5-fold (p b 0.05) and aaDCs by 13-fold (p b 0.001) (Fig. 1B). Likewise, lupus vdDCs (by 2-fold) (p b 0.05) and aaDCs (by 10-fold) (p b 0.001) also expressed significantly lower CD83 than matDCs. Compared with matDCs, normal aaDCs expressed significantly lower CD40, CD86 and CD80 (all p b 0.05) whereas lupus aaDCs showed lower CD40 (p b 0.001) and CD86 (p b 0.05) but not CD80. Normal vdDCs expressed significantly lower CD86 and CD80 (both p b 0.05) than matDCs whereas lupus vdDCs had lower CD40 (p b 0.001). These findings revealed semi-mature phenotype in lupus aaDCs and vdDCs.

3.2. Lupus aaDCs had stable phenotype despite immunostimulatory challenges To study the stability of this semi-mature phenotype, lupus aaDCs and vdDCs were cultured in the presence of sCD40L, toll-like receptor (TLR) agonists, IFN-α, inactive and active lupus serum. sCD40L provides signal for DC activation simulating CD40-CD40L interaction with activated T cells [17]. MDDCs express a variety of TLRs including TLR7 [18] and TLR9 [19] that are predominantly expressed by plasmacytoid DCs. TLR7, TLR9 and IFN-α play important role in the pathogenesis of SLE [20]. R848 is an imidazoquinoline compound that activates DCs via TLR7/TLR8 [21]. CpG-DNA binding to TLR9 activates plasmacytoid DCs and IFN-α production [22]. IFN-α and other soluble factors in active SLE serum promote overactive phenotype of peripheral monocytes [12,22,23]. The levels of costimulatory molecules on lupus aaDCs were not affected by these immunostimulants (Fig. 2A) and remained significantly lower than matDCs (all p b 0.05) (Fig. 2B). Challenge of lupus aaDCs and vdDCs by sera from SLE patients with active disease (SLEDAI mean + standard deviation 16.2 + 9.6, range 6–16) did not affect their significantly low levels of co-stimulatory molecules (Fig. 2C). Furthermore, expression of co-stimulatory molecules remained low in lupus aaDCs challenged by IFN-α but there was no significant upregulation of maturation markers of all MDDCs in response to R848 (Fig. 2D). Thus, lupus aaDCs acquired a stable maturation-resistant phenotype.

2.9. Statistical analysis Statistical analysis was performed by SPSS16.0 (Chicago, USA). Categorical data are reported as mean ± standard error of the mean (SEM) unless otherwise specified. Non-parametric tests were used to analyse differences between groups for

3.3. Lupus aaDCs but not vdDCs attenuated proliferation of lupus memory T cells We next examined if lupus aaDCs had tolerogenic function by studying their suppressive effect on activation and proliferation

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A

B

Figure 1 Alternatively activated dendritic cells generated from lupus and healthy MDDCs by vitD3 and dexamethasone acquired semi-mature phenotype. Monocyte-derived DCs (MDDCs) were generated from CD14 + monocytes from SLE patients and healthy subjects in culture with IL-4 and GM-CSF. On day 8, mature DCs (matDCs) were generated by addition of LPS for 24 h, vdDCs were generated by addition of vitD3 for 24 h in the presence of LPS and alternatively activated DCs (aaDCs) were generated by treating DCs with dexamethasone on day 5, followed by addition of dexamethasone, vitD3 and LPS on day 8 for 24 h. (A) Expression of activation and maturation markers including HLA-DR, CD83, CD40, CD86 and CD80 on matDCs, vdDCs and aaDCs. Flow diagram shows data from one representative experiment. Shadow: isotypic control; dotted line: matDCs; dash line: vdDCs; solid line: aaDCs. (B). Mean fluorescence intensity (MFI) of activation and maturation markers on matDCs, vdDCs and aaDCs derived from SLE and healthy MDDCs. Data was obtained from over 8 patients and is presented as mean ± SEM. *p b 0.05, #p b 0.001.

aaDCs derived from SLE patients have tolerogenic phenotype and function of allogeneic T cells primed by matDCs (Tmat), vdDCs (Tvd) and aaDCs (Taa) from SLE patients and healthy subjects. Four co-culture systems were examined: normal DCs with normal T cells, normal DCs with SLE T cells, SLE DCs with normal T cells and SLE DCs with SLE T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, separate experiments were performed to evaluate differential immunomodulatory effects of aaDCs on naïve and memory T cells. For normal DCs in co-culture with normal or SLE T cells, Taa exhibited significantly lower CD25 and CD69 (both p b 0.05) (Fig. 3A) and suppressed proliferation (p b 0.01) compared with Tmat (Fig. 3B). This attenuation in allostimulatory effect

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by aaDCs was observed on both naïve and memory T subsets. Although lupus T cells expressed higher levels of CD25 and CD69 than normal T cells, both responded to normal aaDCs with reduced proliferation in a similar manner (p b 0.01). Normal and lupus Tvd also showed attenuated proliferation at intermediate level (both p b 0.05) although lower CD69 expression (p b 0.05) was found only in lupus Tvd. For SLE DCs in co-culture with normal or SLE T cells, Taa also demonstrated significantly lower CD25 and CD69 (both p b 0.05) and suppressed proliferation (p b 0.01) compared with Tmat and in a similar manner for both naïve and memory T subsets. Normal Tvd from both naïve and memory

A

Figure 2 Lupus aaDCs had stable semi-mature phenotype despite challenges by soluble CD40L, TLR agonists, IFN-α, inactive and active SLE serum. The maturity status of matDCs, vdDCs and aaDCs derived from lupus MDDCs was challenged by immunostimulants relevant to lupus pathogenesis and inactive and active SLE serum. (A) Expression of activation and maturation markers on lupus matDCs, vdDCs and aaDCs challenged by sCD40L, CpG-DNA, and inactive SLE serum (n = 5). Flow diagram shows data from one representative experiment. Shadow: isotypic control; dotted line: matDCs; dash line: vdDCs; solid line: aaDCs. (B) Mean fluorescence intensity of activation and maturation markers on challenged matDCs, vdDCs and aaDCs derived from SLE and healthy MDDCs. (C) Expression of activation and maturation markers on lupus matDCs, vdDCs and aaDCs challenged by active SLE serum (n = 5). (D) Expression of co-stimulatory molecules on lupus matDCs, vdDCs and aaDCs challenged by R848 and IFN-α. Data was obtained from 5 patients and is presented as mean ± SEM.*p b 0.05.

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B

C

D

Figure 2 (continued).

T cell subsets also had attenuated proliferation (p b 0.05). However, despite significantly lower CD25 and CD69 (both p b 0.05) expressed on lupus Tvd derived from memory T cells, lupus Tvd proliferation was not suppressed.

3.4. Lupus aaDCs induced IL-10 producing cells from naïve T cells and downregulated IFN-γand IL-17 production by memory T cells Next, we examined if aaDCs may have differential immunomodulatory effects on the function of allogeneic naïve and memory T cells. Normal aaDCs in co-culture with normal or

lupus naïve T cells induced significantly less T effector cells that produced IFN-γ and IL-17 (both p b 0.05) (Fig. 4). On the other hand, there was increased IL-10 producing T cells compared to Tmat (p b 0.05) although fewer IL-10 producing T cells were induced among lupus (7.2 + 0.6%) than normal Taa (9.3 + 0.7%) (p b 0.05). Cytokine profile of normal Tvd was not different from Tmat whereas fewer IL-17 producing cells were found among lupus Tvd (p b 0.05). Lupus aaDCs also induced fewer IFN-γ and IL-17 but more IL-10 producing cells from normal or lupus naïve T cells (all p b 0.05) but the proportion of IL-10 producing T cells among lupus (6.2 + 0.7%) and normal (10.5 + 0.8%) Taa were not different.

aaDCs derived from SLE patients have tolerogenic phenotype and function

A NC DC+ NC/SLE T cell

49

SLE DC + NC/SLE T cell

B NC DC+ NC/SLE T cell

SLE DC + NC/SLE T cell

Figure 3 Naïve and memory T cells primed by lupus and healthy aaDCs expressed low levels of activation markers and had attenuated proliferation. Tolerogenic function of MDDCs was evaluated by suppressive effect on activation and proliferation of allogeneic T cells primed by matDCs (Tmat), vdDCs (Tvd) and aaDCs (Taa) from SLE patients and healthy subjects. Four co-culture systems were examined: normal DCs with normal T cells, normal DCs with SLE T cells (left side panels), SLE DCs with normal T cells and SLE DCs with SLE T cells (right side panels). Separate experiments were performed to evaluate differential immunomodulatory effects on CD4+CD45RA+ naïve and CD4+CD45RO+ memory T cells. (A) Expression of activation markers including CD25 and CD69 on Tmat, Tvd and Taa. (B) Proliferation of allogeneic naïve or memory T cells in MDDC co-culture after 6 days. Data was obtained from over 9 patients and is presented as mean ± SEM.*p b 0.05, #p b 0.01.

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In the system of normal or lupus DCs in co-culture with memory T cells, there were significantly fewer IFN-γ and IL-17 producing cells among normal or lupus Taa compared

NC DC+ NC/SLE T cell

with Tmat (both p b 0.05) but there was no induction of IL-10 producing cells. Cytokine profile of normal Tvd was not different from Tmat whereas less IFN-γ producing cells were

SLE DC + NC/SLE T cell

Figure 4 Lupus and normal aaDCs polarized naïve T cells into IL-10 producing cells and downregulated IFN-γ and IL-17 production by memory T cells. Intracellular cytokine profile including IFN-γ, IL-4, IL-17 and IL-10 of naïve and memory T cells in co-culture with matDCs, vdDCs and aaDCs was examined after 6 days. Results of the four co-culture systems: normal DCs with normal T cells, normal DCs with SLE T cells (left side panels), SLE DCs with normal T cells and SLE DCs with SLE T cells (right side panels) are shown. Data was obtained from over 9 patients and is presented as mean ± SEM. *p b 0.05.

aaDCs derived from SLE patients have tolerogenic phenotype and function

proliferation comparable to CD4+CD25hi Treg control. Responder normal and lupus T cells showed similar suppressive response to lupus Taa and Tvd. To examine if these T cells with immunoregulatory function possessed phenotype of inducible Treg, mRNA of transcription factors for Th1 (T-bet), Th2 (GATA3), Th17 (RORγt) and Treg (Foxp3) were studied by real-time PCR in MDDC-primed naïve T cells on Day 6. Normal Taa primed by lupus aaDCs expressed significantly lower mRNA levels of T-bet and RORγt but higher Foxp3 compared with normal Tmat (all p b 0.05) (Fig. 5B). SLE Taa also expressed significantly lower RORγt (p b 0.05) but only a trend of higher Foxp3 was observed. At the protein level, intracellular Foxp3 was highly expressed in SLE Tmat and among various MDDCs primed lupus memory T cells (Fig. 5C). Other Treg markers including CTLA4 and GITR were not found to be upregulated in both normal and lupus aaDC-primed T cells (data not shown).

found among lupus memory T cells in response to normal vdDCs (p b 0.05). Table 1 shows a summary of the findings of the differential MDDC effects on allogeneic naïve and memory T cells. In sum, SLE aaDCs were able to suppress activation and proliferation of naïve and memory T cells and were able to polarize naïve T cells into IL-10 producing effector cells and modulate memory T cells to less pro-inflammatory phenotype as normal aaDCs. On the other hand, lupus vdDCs attenuated proliferation of naïve and memory T cells to a lesser extent compared with lupus aaDCs and did not show remarkable modulatory effect on the cytokine profile of these T subsets. Thus, aaDCs generated from combination of vitD3 and dexamethasone appeared to possess more potent tolerogenicity compared with vdDCs generated using vitD3 as single agent in SLE patients. Lupus T cells generally responded to aaDCs in similar manner as normal T cells with the exception of a lower proportion of IL-10 producing cells induced from lupus naïve T cells compared with normal T cells.

3.6. Normal and lupus aaDCs demonstrated unique IL-12p70loIL-10hi cytokine profile

3.5. Lupus aaDCs induced IL-10 producing T cells possessed regulatory function

As DCs are potent antigen presenting cells and the cytokine milieu shaped by DCs determines polarisation of naïve T cells into distinct effector subsets, we studied the cytokine profile of these MDDCs. Normal matDCs produced high IL-12p70 and IL-6 but low IL-10 levels (Fig. 6A) whereas normal aaDCs produced significantly lower IL-6 by 66%, almost no production of IL-12p70 and 2-fold higher IL-10 compared with normal matDCs (all p b 0.05). Although SLE matDCs produced less IL-12p70 and more IL-10 compared to normal matDCs, lupus aaDCs also expressed lower IL-12p70 and IL-6 and higher IL-10

To evaluate whether these IL-10 producing T cells induced by lupus aaDCs from naïve T cells possessed immunoregulatory function, T cell suppression assay was performed by co-culturing responder allogeneic CD4+CD25− T cells depleted for Treg in the presence of irradiated MDDC-primed T cells and graded doses of matDCs obtained from third-party subjects. As expected, increased proliferation was observed in T cells co-cultured with Tmat (Fig. 5A). Normal or lupus Taa and Tvd demonstrated suppressive effect on allogeneic T cell

Table 1

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Summary of differential immunomodulatory effects of aaDCs and vdDCs derived from SLE patients and normal subjects. Naïve

Normal DC Co-stimulatory molecules CD25 CD69 Proliferation Cytokine producing cells IFN-γ IL-4 IL-17 IL-10 SLE DC Co-stimulatory molecules CD25 CD69 Proliferation Cytokine producing cells IFN-γ IL-4 IL-17 IL-10

Memory

Taa

Tvd

Taa

Tvd

↓ ↓ ↓↓

– ↓(lupus T only) ↓

↓ ↓ ↓↓

– ↓(lupus T only) ↓

↓ – ↓ ↑

– – ↓(lupus T only) –

↓ – ↓ –

↓(lupus T only) – – –

Taa

Tvd

Taa

Tvd

↓ ↓ ↓↓

– – ↓(lupus T only)

↓ ↓ ↓↓

↓(lupus T only) ↓(lupus T only) ↓(lupus T only)

↓ – ↓ ↑

– – – –

↓ – ↓ –

– – – –

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production (all p b 0.05). On the other hand, normal vdDCs produced significantly lower IL-12p70 and IL-6 with no increase in IL-10 whereas lupus vdDCs expressed only lower

A

B SLE DC+NC/SLE naive T cell

IL-12p70 compared to matDCs (all p b 0.05). Thus, both normal and lupus aaDCs presented unique cytokine profile of IL-12p70loIL-10hi.

aaDCs derived from SLE patients have tolerogenic phenotype and function

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Figure 5 Naïve T cells primed by lupus aaDCs demonstrated suppressive effect on allogeneic T cell proliferation. (A) Naïve lupus T cells primed by lupus matDCs, vdDCs or aaDCs were irradiated and co-cultured with allogeneic CD4+CD25-responder T cells from SLE or healthy subjects, in the presence of increasing doses of allogeneic normal matDCs. Irradiated CD4+CD25hi cells were used as positive control. Diagram showed proliferation of responder T cells as examined by 3H-thymidine incorporation after 5 days. (B) mRNA levels of transcription factors of T effector cells including T-bet, GATA-3, RORγt and Foxp3 were measured from SLE or healthy naïve T cells in co-culture with lupus matDCs, vdDCs and aaDCs for 6 days and are expressed as relative expression normalized to ß-actin. (C) Percentage of intracellular Foxp3 expressing cells among Tmat, Tvd, Taa in naïve and memory T cell subsets in response to lupus MDDCs on day 6. Data was obtained from over 5 patients and is presented as mean ± SEM. *p b 0.05.

3.7. Possible mechanisms underlying tolerogenicity in lupus aaDCs Next, we examined mechanisms underlying tolerogenicity of aaDCs. As apoptotic DC uptake was found to convert immature DC to tolerogenic DC [24], we studied proportion of Annexin-V positive MDDCs and T cells from lupus patients and healthy subjects in various culture conditions. Apoptosis of lupus and normal aaDCs and Taa were not found to be increased (data not shown). Although aaDCs showed unique IL-12p70loIL-10hi cytokine profile, neither rIL-12 nor neutralizing anti-IL-10 antibody reversed the suppressive effect of aaDCs on CD25 expression and allogeneic T cell proliferation (Fig. 6B), suggesting that these cytokines may not play a significant role in the mediation of aaDC tolerogenicity. Indoleamine 2, 3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, was previously shown to mediate tolerogenicity in some DCs [25]. However, IDO mRNA expression was not found to be different between aaDCs and matDCs from SLE patients or healthy subjects (Fig. 6C). We then examined expression of RelB, a transcription factor required for DC maturation [26], and found significant reduction in expression of RelB mRNA (Fig. 6D) and protein (Fig. 6E) in lupus aaDCs and vdDCs and in normal aaDCs compared with matDCs (both p b 0.05).

4. Discussion In this study, we generated aaDCs from SLE patients with quiescent disease by treating peripheral monocytes in vitro with vitD3 and dexamethasone followed by LPS-induced maturation. aaDCs derived from pharmacological agents such as vitD3 or dexamethasone as single agent [27–29] or in combination [30,31] after LPS-induced maturation has

previously been shown to acquire stable semi-mature and tolerogenic phenotype and can prolong organ allograft survival [32]. LPS treatment in aaDC generation is essential for its regulatory function and migratory capability [33]. While mature DCs showed potent Th1-driving phenotype with predominant production of IL-12 [34], we found that lupus aaDCs expressed high level of MHC Class II but low co-stimulatory molecules and maturation markers. Like aaDCs derived from healthy subjects, lupus aaDCs attenuated allogeneic T cell activation and proliferation and presented cytokine profile of IL-12p70loIL-10hi as well as low IL-6 production. This semi-mature phenotype of lupus aaDCs remained stable upon challenges by sCD40L, TLR receptor agonists, IFN-α, inactive and active lupus serum. We demonstrated that lupus aaDCs had differential immunomodulatory effects on naïve and memory T cells. In response to aaDCs, naïve T cells were polarized into IL-10 producing Treg whereas memory T cells were modulated towards a less pro-inflammatory phenotype with suppressed IFN-γ and IL-17 production. The lack of IL-10 induction in memory T cells was likely related to their terminally differentiated status as lupus memory T cells expressed high IL-10 de novo and there was no further increase in IL-4 production with MDDC co-culture to suggest a skew to Th2 profile. Both IL-10 producing T cells derived from healthy subjects and SLE patients were shown to suppress allogeneic T cell activation and proliferation. Furthermore, the low-level IL-6 produced by aaDCs favoured differentiation of Treg over Th17 [35]. These IL-10 producing T cells with regulatory function were compatible with type 1 Treg (Tr-1) which have previously been shown to be induced by vitD3 and dexamethasone generated aaDCs [26–31]. These Tr-1 cells were characterised by production of high level IL-10 with immunoregulatory function but unlike natural Treg that develop from thymus, Tr-1 cells may not express Foxp3. Nevertheless,

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Foxp3 in human is not specific for Treg phenotype and is also expressed by activated T cells [36] as reflected by high Foxp3 expression in lupus memory T cells in our study.

Manipulation of IL-12 and IL-10 level in lupus aaDC-T cell co-culture by exogenous rIL-12 or neutralizing IL-10 treatment did not reveal remarkable role of these cytokines in

A

B NC DC+NC naïve T cell

C

SLE DC+NC naïve T cell

D

Figure 6 Mechanisms underlying tolerogenicity of lupus and normal aaDCs. (A) Cytokine levels including IL-6, IL-12p70 and IL-10 were measured in supernatant from matDCs, vdDCs and aaDCs culture by ELISA. (B) CD25 expression and proliferation were examined on primed naïve T cells in the presence of recombinant IL-12, neutralizing anti-IL-10 antibody and IgG2b isotypic control antibody in DC-T cultures. Data was obtained from 4 experiments and is presented as mean ± SEM. (C) mRNA expression of IDO and (D) RelB relative to β-actin in matDCs, vdDCs and aaDCs from SLE patients and healthy subjects was examined. (E) Western blot revealed 68 kD band corresponding to RelB protein with quantitative measurement by densitometry. Data was obtained from over 5 patients and is presented as mean ± SEM.* p b 0.05.

aaDCs derived from SLE patients have tolerogenic phenotype and function

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E

Figure 6 (continued).

the tolerogenicity of aaDCs despite one study that showed low-level IL-12 produced by aaDCs was functionally related to hyporesponsive memory T cells [10]. Instead, IL-10 produced by tolDCs was suggested to be crucial in the maintenance of their stable maturation-resistance properties as neutralization of IL-10 in tolDC generation caused partial DC maturation [29]. Our finding of attenuated pro-inflammatory cytokine production from memory T cells may also be a result of induction of T cell anergy via low-level costimulatory molecules expressed on aaDCs in cognate interaction with these memory T cells [37]. Uptake of apoptotic cells by immature DCs was shown to be pro-tolerogenic [38,39] and is relevant to SLE which is characterised by increased immune cell apoptosis [40]. In our study, increased apoptosis of aaDCs or Taa was not observed. Expression of inhibitory molecules or enzymes such as PDL-1 [27,41] and IDO [25] has also been shown to mediate tolerogenicity in DCs but we did not show any difference in IDO expression. On the other hand, lupus and normal aaDCs were found to express low RelB transcription. RelB is a transcription factor involved in non-canonical NF-κB pathway and is a key regulator of differentiation and maturation of DCs [26,42]. Indeed, vitD3 was shown to repress transcription of RelB promoter activity in DCs with inhibition of NF-kB translocation [43]. Although the low RelB expression in aaDCs compared with mature DCs demonstrated in our study may only reflect the maturation status of these DCs, RelB silenced DCs were previously demonstrated to have immunosuppressive function in vitro [44]. Furthermore, in vivo adoptive transfer of RelB-silenced DCs can improve survival in allogeneic heart transplantation [44] and alleviate symptoms in experimental autoimmune myasthenia gravis [45]. Thus, the role of low-level RelB expression as a key mediator in induction of tolerogenicity of aaDCs warrants further study. Beneficial therapeutic effects of vitD3 and dexamethasone generated aaDCs have previously been demonstrated in antigen-specific system such as collagen-induced arthritis [46]. We demonstrated suppressive effect of Taa on third-party T cell responders which may be related to high-level IL-10 produced by these cells that contributes to broad non-antigen specific anti-inflammatory effect [47]. This may be of therapeutic benefit in non-organ specific autoimmune diseases like SLE where a diversity of unknown autoantigens may play a role in the pathogenesis. Furthermore, tolDCs generated in the absence of pulsed specific

peptides were shown to elicit robust proliferation of polyclonal Treg [48]. Unpulsed autologous tolDCs of allograft recipients were found to induce donor-specific tolerance in MHC-mismatched rat cardiac allograft [49]. These tolDCs co-localised with leukocytes expressing donor antigens in the spleen and may capture and process donor antigens in vivo [49]. Previous study used non-pharmacological means to induce tolDCs in SLE patients by co-culturing lupus MDDCs with iC3b-opsonized apoptotic cells but was effective in only one-third of lupus patients [50]. On the other hand, lupus MDDCs in patients receiving vitamin D supplement was found to express lower IFN-α genes in response to a challenge by active SLE plasma [51]. This is the first time that combination of vitD3 and dexamethasone is applied in the generation of tolDCs in the lupus setting. Our data showed that lupus aaDCs generated in this manner have comparable efficacy to normal aaDCs and superior tolerogenicity over using vitD3 as single agent. Lupus vdDCs were less efficient in the attenuation of proliferation and pro-inflammatory cytokine production by naïve and memory T cells. Whether this deficiency may be related to aberrant vitD receptor expression and function in lupus MDDCs warrants further study, as vitD receptor polymorphism has been linked to increased risk of SLE [52]. Indeed, maturation-resistant tolDCs generated by combination of vitD3 and dexamethasone have previously been shown to have superior tolerogenic profile over individual agents [53] as well as other pharmacological agents [30]. A recent paper reported that these aaDCs could also induce regulatory B cells [30]. Thus, combination of vitD3 and dexamethasone followed by LPS maturation represents an effective protocol in the induction of tolDCs in SLE patients with therapeutic implications. Our study also gives clue to the observed activated phenotype of peripheral monocytes in lupus. The variations in efficiency in the generation of tolDCs from lupus patients as demonstrated in our study and Berkun et al. [50] suggested that MDDCs from lupus patients are highly susceptible to extrinsic influence such as cellular factors and pharmacological agents. However, we showed that it is feasible to generate aaDCs from SLE patients with quiescent disease that demonstrate comparable phenotype and function to those derived from healthy subjects. Combination of vitD3 and dexamethasone followed by LPS maturation likely represented a superior method over vitD3 as single agent in the induction of tolDCs in SLE patients. In regard to generation of tolDCs from SLE patients with active disease, our preliminary data

56 showed that aaDCs generated from active and inactive SLE patients did not differ in terms of co-stimulatory molecule expression and production of IL-6 and IL-12 (result not shown). Further characterization of full profile of aaDCs generated from active SLE patients is warranted with therapeutic implication of applying autologous tolDCs as cell-based therapy in the restoration of immune tolerance in this condition. SLE T cells were previously found to be resistant to induction of anergy in murine lupus model [54] and reduced number or defective function of Treg were reported in lupus patients [55]. Our data suggested that lupus T cells may be slightly deficient in differentiation into IL-10 producing T cells induced by normal aaDCs compared with naïve T cells from healthy subjects although these aaDC primed-naïve lupus T cells have comparable function in attenuation of allogeneic T cell activation and proliferation. We cannot exclude small amount of effector cell contamination related to isolation kit efficiency and indeed, peripheral T cells in SLE patients were described to be dominated by terminally differentiated memory T cells with limited proliferative capability [56]. Given the important role of memory T cells in lupus pathogenesis, we demonstrated a beneficial immunomodulatory role of aaDCs on lupus memory T cells towards less pro-inflammatory profile with reduced Th1 and Th17 cytokine production.

5. Conclusions In conclusion, we showed that aaDCs generated from SLE patients with quiescent disease by treatment of vitD3 and dexamethasone had comparable tolerogenicity as normal aaDCs and superior tolerogenic profile over vitD3 alone. These aaDCs have potential therapeutic advantages in SLE as they can induce IL-10 producing Tr-1 cells from naïve T cells and modulate cytokine production towards a less pro-inflammatory profile with suppressed proliferation in memory T cells. Thus, vitD3 and dexamethasone induced aaDCs may emerge as potential cell-based therapy in this disease.

Conflict of interest statement The author(s) declare that there are no conflicts of interest.

Authorship MY Mok, CS Lau, L Lu – Study design and supervision; MY Mok, Y Lo – Patient recruitment; HJ Wu, D Luk – Experiments.

Acknowledgments This study was supported by a university research grant of the University of Hong Kong.

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