Functional significance of MAIT cells in psoriatic arthritis

Cytokine 125 (2020) 154855

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Functional significance of MAIT cells in psoriatic arthritis a

a

a

Smriti K. Raychaudhuri , Christine Abria , Anupam Mitra , Siba P. Raychaudhuri a b

a,b,⁎

T

VA Medical Center Sacramento, CA, USA Division of Rheumatology, Allergy & Clinical Immunology, University of California Davis, School of Medicine Sacramento, CA, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: MAIT cells Psoriatic arthritis IL-23R IL-17A

Background: Mucosal-associated invariant T (MAIT) cells are gaining more relevance for autoimmune diseases because of its (i) innate and adaptive immune response (ii) tissue homing properties (iii) production of IL-17A. These cells are predominantly CD8+ cells, because of its strong association with MHC-I. Tc17 CD8+/MAIT cells likely to have a critical role in psoriatic arthritis (PsA). Herein, we have explored pathological significance of MAIT cell in PsA. Methods: Peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) were collected from age/sex matched (n = 10 for each) PsA, rheumatoid arthritis (RA) and osteoarthritis patients (OA). Hi-D FACS studies were performed: (i) activated memory cells (CD3+CD45RO+) T cells were identified (ii) gating strategies were made to identity the MAIT (CD3+Vα7.2TCR+CD161hi) cells, its phenotype pattern; and functional significance in respect to IL-17A production and responsiveness to human rIL-23. Anti CD3/CD28 ab cocktail was used to activate cells along with rIL-23 to culture and enrich the MAIT cells. The percentages of each cell population and the mean fluorescence intensity (MFI) were analyzed using Flow Jo software. Results: MAIT cells were enriched in synovial fluid of PsA (4.29 ± 0.82%) compared to PBMC (1.04 ± 0.71). With stimulation, SFMC MAIT cells produced significantly more IL-17A (32.66 ± 4.01%) compared to that of RA (23.93 ± 2.81%, p < 0.05) and OA (5.02 ± 0.16%, p < 0.05). MAIT cells were predominantly CD8+ (> 80%). Significant upregulation of IL-23R was noted in synovial fluid MAIT cells of PsA (24.97 ± 2.33%, p < 0.001) and RA (21.93 ± 2.29%, p < 0.001) compared to that of OA (2.13 ± 2.29). This IL-23R was functionally active as evidenced by profound mitotic effect in presence of rIL-23. Conclusion: MAIT cells are poly functional; produce multiple cytokines (IL-17A, IFN-γ, TNF-α). Here, we demonstrated synovial fluid MAIT cells as a major source of IL-17A and majority of MAIT cells were CD8+. Functionally active IL-23R on these migrated MAIT cells brings a new dimension. They may not need MR1 associated activation rather lesional IL-23 in the synovium can independently regulate these critical Tc17 CD8+ MAIT cells. Thus, these cells likely to be a part of the IL-23/IL-17A cytokine network and play a critical role in the pathogenesis of PsA.

1. Introduction Psoriatic arthritis (PsA) is a multifactorial inflammatory arthritis, commonly associated with psoriasis and predominantly affecting ~30% of patients with psoriasis [1,2]. The exact cause of PsA is unknown, however, it has been shown that the interplay between genetic, environmental and immunological factors significantly contributes in its pathogenesis [3,2]. Studies have shown abundance of IL-17A producing T-cells at the disease site such as in skin and joint in patients with

psoriasis and PsA respectively [4–6]. IL-17A has been shown to induce production of pro-inflammatory cytokines and chemokines such as IL-6, IL-8, CCL20; IL-17A can perpetuate the inflammatory process and has osteoclastogenic effect [4,6,7]. Further, success of clinical studies targeting IL-17A pathway have confirmed its critical role in the pathogenesis of psoriasis and PsA [8–10]. CD4+ and CD8+ T cells are major source of IL-17A in skin and joint tissue [6,4]. Genetic studies of psoriatic disease have shown various susceptibility loci in Th17 pathway (IL-23A, IL-23R), pathway of innate immunity and CD8 T cells

Abbreviations: FSC, forward scatter; MAIT, Mucosal-associated invariant T; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; OA, osteoarthritis; PsA, psoriatic arthritis; qPCR, quantitative real-time polymerase chain reaction; RA, rheumatoid arthritis; SF, synovial fluid; SFMC, synovial fluid mononuclear cells; SSC, side scatter; TCR, T cell receptor ⁎ Corresponding author at: Division of Rheumatology, Allergy & Clinical Immunology, University of California Davis, School of Medicine, 10535 Hospital Way, Bldg # 807, Mather, CA 95655, USA. E-mail address: [email protected] (S.P. Raychaudhuri). https://doi.org/10.1016/j.cyto.2019.154855 Received 26 February 2019; Received in revised form 8 September 2019; Accepted 9 September 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.

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(ZAP70, ERAP1) [11,12]. In psoriasis, epitropism of CD8+ T cells and oligoclonal expansion of these cells have been shown [13,5,14]. CD8+ T cells recognize antigens presented by MHC class I molecule. Both psoriasis and PsA are associated with HLA class [3,15,12,16]. Mucosal-associated invariant T (MAIT) cells are innate-type T cells with restricted T cell receptor (TCR) diversity. MAIT cells are activated by recognizing an MHC class I like molecule - MR1, a vitamin metabolite produced by bacteria or yeast [17]. These cells are predominantly developed in gut mucosa in presence of commensal flora as evidenced by lack of MAIT cells in germ-free mice [18]. Being a part of innate immunity, MAIT cells mount rapid immune response to bacteria or yeast infection compared to conventional CD8+ T cells [7,18,19]. These cells are in abundance (1–10%) in blood, liver, and gut, and produce IL-17A, TNF-α and IFN-γ on activation [7,19,20]. MAIT cells are identified by CD3+CD8+ cells expressing TCR Vα7.2 TCR, CD161hi, CCR6, and RORγt (Th17 cells transcription factor) [7,20]. Apart from its protective role in bacterial infection, recent studies have suggested pathogenic role of MAIT cells in different autoimmune diseases [21–25]. In this context, a recent study by Gracey et al. showed decreased frequency of IL-17A+ MAIT cells in blood whereas increased frequency of these cells in synovial fluid (SF) of ankylosing spondylitis (AS) patients compared to healthy controls [24]. In another study, Teunissen et al. demonstrated higher frequency of IL-17A producing MAIT cells and conventional IL-17A producing CD8+ T cells (Tc17) in skin particularly in epidermis which further suggest pathogenic role of MAIT cells in psoriasis [25]. A study in psoriatic arthritis patients showed increased numbers of IL-17+CD3+CD8+ T cells in SF of PsA patients compared to paired peripheral blood and SF of RA patients, and its positive correlation with disease severity [26]. Among these IL17+CD3+CD8+ T cells, they also could identify certain MAIT cell variant but did not further characterize nor investigated their functional significance. These findings are similar to what other studies have found in psoriatic skin plaques- dominance of CD8+ T cells [5,14,27]. These recent findings as well as strong association of PsA with MHC class I suggest crucial pathogenic role of CD8+ T cells in pathogenesis of PsA. MAIT cells have been implicated in autoimmune diseases. Murine model experiments as well suggest that MAIT cells can have a critical role in autoimmune arthritis. Altogether this T cell subpopulation characterizes all the potentials for a contributing role in the inflammatory-proliferative cascades of PsA. Here we have elucidated the relevance and significance of the MAIT cell in PsA.

Table 1 Demographical and clinical characteristics of the study cohorts. Data are expressed as mean ± standard deviation unless otherwise specified. Demographic and clinical data

PsA (n = 10)

RA (n = 10)

OA (n = 10)

Male sex (N/%) Age (years) Disease duration (Years) ESR (mm/h) CRP (mg/L) N. of tender joints N. of swollen joints Pain VAS BSA DAS28 (ESR) RF positivity csDMARDs (in last 3 months) bDMARDs (in last 3 months)

7(70) 54.5 ± 11 11.8 ± 5 16.6 ± 6.2 8 ± 4.2 5.2 ± 2.1 3.4 ± 1.5 5.75 ± 3.2 8.2 ± 4.4 4.5 ± 0.8 0 (0) 0 (0) 0 (0)

8(80) 52.1 ± 10 15.5 ± 8.2 24 ± 6.2 14.2 ± 2.6 6.8 ± 3.1 5.2 ± 2.2 6.2 ± 1.8

8(10) 55.5 ± 10 15 ± 10.2 6 ± 2.2 2 ± 1.2

5.1 ± 1.1 10 (0) 0 (0) 0 (0)

5.2 ± 1.8

0 (0) 0 (0) 0 (0)

PsA, Psoriatic Arthritis; RA, Rheumatoid arthritis; OA, Osteoarthritis; DAS28, Disease Activity Score 28; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, Rheumatoid Factor; VAS, visual analoghe scale; csDMARDs, conventional synthetic Disease-modifying antirheumatic drugs; bDMARDs, biological Disease-modifying antirheumatic drugs.

azathioprime and biologic agents within last 3 months, and who did not receive any topical treatment for psoriasis other than emollients in the past 4 weeks. Patients had complete physical examination, evaluation for severity of psoriasis and arthritis, appropriate blood tests, and radiological studies. Psoriatic arthritis patients had either oligoarthritis or symmetric polyarthritis. Synovial fluid of knee was collected for therapeutic intervention or before injecting cortisone in the joint. 2.2. T Cell isolation & stimulation Peripheral blood mononuclear cells (PBMCs) were purified from venous blood collected by sodium citrate vacutainer using Lymphocyte Separation Medium (MP Biomedicals, LLC) by density-centrifugation as described in earlier studies [6,28]. Synovial fluid acquired by joint aspiration was incubated with hyaluronidase enzyme (1 mg/mL) in phosphate buffered saline for 30 min. Synovial fluid mononuclear cells (SFMCs) were then isolated by density-centrifugation using Lymphocyte Separation Medium. PBMCs and SFMCs cell viability was confirmed by Trypan Blue exclusion (> 90% viable). PBMCs and SFMCs were washed and resuspended in RPMI media (Cellgro) with 10% fetal bovine serum, 2 mM glutamine, penicillin/streptomycin and 20 mM HEPES. Cells were seeded in 24-well plates at 1 × 106 per well. Cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1 μg/mL ionomycin (Sigma-Aldrich) for 4 h. Alternatively, 1 × 106 PBMCs or SFMCs were incubated for 6 days on 24well plates pre-coated with 5 μg/mL anti-CD3 (UCHT1, eBioscience) and with 2 μg/mL soluble anti-CD28 (CD28.2, eBioscience) and 40 ng/ mL recombinant IL-23 for 3 days. After 3 days cells were washed and resuspended in 1 ml of culture medium and 40 ng/mL recombinant IL23 [29]. On the day 6, cells were stimulated with 50 ng/mL PMA, 1 μg/ mL ionomycin. Monensin (2 μM, Sigma) was added to cell culture for intracellular staining.

2. Materials and methods 2.1. Subjects PsA patients who fulfilled the CASPAR (classification criteria for Psoriatic Arthritis) criteria were recruited in this study. OA of the knee and RA were diagnosed according to the clinical, laboratory, radiographic criteria of the American College of Rheumatology. Patients were recruited from the Rheumatology clinic at Sacramento VA Medical Center, CA, USA. After obtaining IRB approved informed consent, peripheral blood and synovial fluid was collected from age and sex matched patients with PsA (n = 10), RA (n = 10) and knee OA (n = 10). Demographical and clinical characteristics of the study cohorts are provided in the Table 1. At the time of enrolment all had active disease; either they were a new consult or had disease flare because of discontinuation of their DMARDs (> 6 ± 2 months). Active PsA or RA was defined by the presence of at least 3 swollen and 3 tender joints. In patients with PsA, in addition to the above, presence of plaque psoriasis with a qualifying lesion of at least 2 cm in diameter was required. All patients were evaluated for the swollen joint/tender joint count, patient’s assessment of pain, ESR and CRP. We enrolled patients who did not take methotrexate, leflunomide, cyclosporine, hydroxychloroquine, sulfasalazine,

2.3. Flow cytometry analysis and intracellular cytokine staining PBMCs and SFMCs were stained with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD161-PE (clone HP-3G10, eBioscience), anti-Vα7.2TCRAPC (clone 3c10, BioLegend), anti-human gamma delta TCR-PE (clone B1.1, eBioscience), anti-αβTCR-APC (clone IP26, eBioscience), antiCD3-BV421 (OKT3, eBioscience), anti-CD3-BV605 (clone SK7, BD Bioscience), anti-CD3-PE-Cy5 (clone SK7, BD Bioscience), antiCD45RO-PE-Cy5 (Clone UCHL1, eBioscience), anti-CD45RO-BV421 (Clone UCHL1, eBioscience), anti-CD4-APC-Cy7 (clone RPA-T4, 2

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A PsA PBMC 17.2%

1.04%

79.6% 1.18%

OA PBMC 3.62%

16.1%

PsA

RA

C

OA 2.96%

4.29%

78.5%

% MAIT cells in T cells

*

1.23%

CD161

*

*

*

8

SFMC

6

1.04%

Percentage

B

1.28%

3.62%

1.29%

4

2

PBMC 0

Ps

VD7.2TCR

A

PB

M

C Ps

A

SF

M

C R

A

PB

M

C R

A

SF

M

C O

A

PB

M

C O

A

SF

M

C

Fig. 1. Frequency of MAIT cells in blood and synovial fluid. Synovial fluid (SF) and peripheral blood (PB) were collected from psoriatic arthritis (PsA, n = 10), rheumatoid arthritis (RA, n = 10) and osteoarthritis (OA, n = 10) patients. MAIT cells were identified as CD3+CD161+Vα7.2TCR+ cells, γδ-T cells as CD3+ γδTCR+, NK cells as CD3+CD161+ and αβ-T cells as αβTCR+ by flow cytometry. (A) Representative histogram showing frequency of MAIT cells, NKT cells, γδ T cells, and αβ T cells in PB of PsA and OA patients. (B) Representative histogram comparing frequency of MAIT cells in paired samples (PB and SF) of PsA, RA and OA patients. (C) Percentage MAIT cells; shown are average values of individual PB and SF samples, n = 10 per patient type. All experiments were done in triplicate. Results were expressed as Mean ± SEM. One-way ANOVA with Tukey multiple comparison test was used to determine the statistical significance. *p < 0.0001, SFMC of PsA and RA compared to SFMC of OA. *p < 0.0001, PBMC of PsA and RA compared to PBMC of OA.

eBioscience), anti-CD8-PE-Cy7 (clone RPA-T8, eBioscience), fluorescein conjugated anti-IL-23R (Clone 218213, R&D Systems). Appropriately conjugated IgG antibodies were used as isotype controls and fluorescence minus one control (FMO) were performed for proper gating. For intracellular staining, following surface staining, cells were fixed and permeabilized using Perm/Fix solution (BD Bioscience) according to the manufacturer’s instructions [6]. Cells were stained with FITC-labelled anti-IL-17A (clone eBio64DEC17, eBioscience). Stained cells were acquired on BD FACS ARIA Fusion flow cytometer. At least 500,000 events for each sample were acquired. Data was analyzed

using FlowJo Version 10 (Treestar). Viable cells were gated by exclusion of cells stained with Live/Dead stain (Invitrogen). Briefly, first we gated lymphocytes based on forward (FSC) and side scatter (SSC) and then selected live cells using live/dead stain. Then CD3+ cells were gated among the live cell population. Then we looked for MAIT cells among these CD3+ T cells using Vα7.2TCR+ vs CD161hi [24,30]. Among these MAIT cells, we then looked for CD4+ and CD8+ cells followed by CD45RO+ (memory cells) cells among these CD4+ and CD8+ MAIT cells.

3

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Fig. 2. Synovial fluid MAIT cells phenotype in PsA patients. Synovial fluid (SF) was collected from psoriatic arthritis (PsA, n = 10) patients. Lymphocytes were gated first based on FSC and SSC, then CD3+ T cells were gated. MAIT cells were identified as CD3+CD161+Vα7.2TCR+ cells among the gated CD3+ T cells. Among this MAIT cells, CD4+ and CD8+ frequencies were seen and finally the memory phenotypes of these cells were identified. Representative FACS plots showing the frequency of MAIT cells and its phenotypes in patients with PsA.

2.4. CfSE

3. Results

As described earlier [28,31], PBMCs and SFMCs were stained with CFSE and stimulated for 6 days with CD3, CD28, recombinant IL-23, PMA, Ionomycin as described above. Cells were then surface stained with respective antibodies described above and acquired on BD FACS ARIA Fusion.

3.1. MAIT cell frequency in paired PBMC and SFMC of patients with PsA, RA and OA: Frequency MAIT cells is higher in patients with PsA and RA First, the frequency of MAIT cells, γδ T cells, NKT cells and αβ-T cells in PBMCs of PsA, RA and OA patients was determined using flow cytometry. MAIT cells were identified as CD3+CD161+Vα7.2TCR+ cells, γδ-T cells as CD3+ γδTCR+, NKT cells as CD3+CD161+ and αβ-T cells as αβTCR+. NKT cells (CD3+CD161+) were further gated for TCR Vα24-Jα18 (iNKT cell) ab positivity. In Fig. 1A, representative FACS plots from PBMC of a PsA and an OA patient are demonstrated for MAIT cells, γδ-T cells, NKT cells and αβ-T cells. Among peripheral blood T cells (CD3+) of PsA patients, frequency of MAIT cells, γδ-T cells, NKT cells and αβ-T cells were 1.2 ± 0.13, 1.8 ± 0.22, 14.5 ± 1.3, 81.5 ± 2.8 respectively. The frequency of MAIT cells in peripheral blood was significantly less in patients with PsA (1.04 ± 0.71) and RA (1.29 ± 0.27) compared to OA (3.62 ± 0.13, p < 0.01). Paired SFMCs were used to determine the frequency of MAIT cells at disease site. In contrast to PBMC findings, frequency of MAIT cells in SFMCs were significantly higher in PsA (4.29 ± 0.82) and RA (2.96 ± 0.31) patients compared to OA (1.23 ± 0.12) (p < 0.0001) (Fig. 1B, C). We did not notice any correlation of MAIT cell numbers/percentage in the synovial fluid with patient’s disease severity.

2.5. Western blot analysis of IL-23R expression in PBMC PBMCs were stimulated for 6 days with CD3, CD28, rIL-23, PMA, Ionomycin as described above. MAIT PBMCs were isolated by magnetic sorting with biotinylated anti-Vα7.2TCR (3C10) and anti-biotin magnetic beads as per manufacture’s protocol (Miltenyi Biotec). The positive selected cells and negative selected cells were lysed and IL-23R was identified by western blot as per our standardized protocol [6,32,33]. Primary antibodies IL-23R (3D7, Santa Cruz Biotechnology) at 1:200 and α-tubulin (CST, Danvers, MA) at 1:1000 dilutions in 5% BSA-TBST were used. Band density was measured using Image J software (NIH, Bethesda, MD, USA). Results are expressed as relative intensity (R.I: intensity of each band adjusted to α-tubulin).

2.6. Quantitative real-time polymerase chain reaction (qPCR) for IL-23R gene expression 3.2. MAIT cell phenotype PBMCs were stimulated for 6 days with CD3, CD28, recombinant IL23, PMA, Ionomycin as described above. MAIT PBMCs were isolated by magnetic sorting with biotinylated anti-Vα7.2TCR (3C10) and antibiotin magnetic beads according to the manufacture’s protocol (Miltenyi Biotec). RNA was extracted from positive and negative selected cells and IL-23R gene expressed was identified by qPCR as per our standardized protocol [32]. Primer pair against IL-23R (Origene, Rockville, MD) and primer pair tata box (Real Time Primers, LLC) were used to evaluated gene expression. Analysis of relative gene expression data normalized to endogenous control was calculated using RQ = 2−ΔΔCT method.

After finding significantly increased frequency of MAIT cells in the local disease site (SFMC) of PsA patients, further phenotype of these cells was determined by staining for CD4, CD8 and CD45RO (Fig. 2). Among MAIT cells of synovial fluid, majority were CD8+ (81.49 ± 2.15%) cells, few CD4+ (4.97 ± 0.55%) cells and remaining cells were CD4−CD8− (12.54 ± 2.65), suggesting NKT cells (Table 2). CD45RO was used to further characterize naïve/memory subtype of these cells. Majority of CD4+MAIT cells (92.5% ± 1.88) and CD8+MAIT cells (84.8% ± 2.36) were CD45RO+ suggesting an effector memory phenotype.

2.7. Statistics

Table 2 . Synovial fluid MAIT (Vα7.2TCR+CD161+CD3+) cells phenotypes in Psoriatic (PsA), Rheumatoid (RA) and Osteoarthritis (OA) patients. Data are expressed as mean ± standard deviation.

All experiments were done in triplicate and results are expressed as Mean ± SEM (Standard Error of Mean). Statistical analysis was done using the GraphPad Prism software, version 8 (GraphPad Software Inc.). Nonparametric unpaired tests (Mann-Whitney U Test) were used to determine statistical significance. Comparisons of more than two groups were done using one-way ANOVA with Tukey multiple comparison test. A p value of < 0.05 was considered statistically significant.

CD4+ CD8+ CD4−CD8−

4

PsA (n = 10)

RA (n = 10)

OA (n = 10)

4.97 ± 0.55% 81.49 ± 1.93% 12.54 ± 2.65%

4.10 ± 0.66% 80.27 ± 2.15% 14.64 ± 3.37%

4.54 ± 0.58% 65.76 ± 2.74% 28.7 ± 1.40%

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Fig. 3. Differential IL-17A production by stimulated MAIT cells in blood and synovial fluid. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated 3d CD3/CD28+rIL-23 followed by 3d rIL-23. (A) Representative FACS plots comparing percentage of MAIT cells (CD3+CD161hiVα7.2TCR+) producing IL-17A by paired PBMC and SFMC of patients with PsA, RA and OA. (B) Percentage of IL-17A+MAIT cells; shown are average values of individual PB and SF samples, n = 10 per patient type. Each patient sample was done in triplicate. Results were expressed as Mean ± SEM. One-way ANOVA with Tukey multiple comparison test was used to determine statistical significance. *p < 0.001, PBMC of PsA and RA compared to PBMC of OA. **p < 0.0001, SFMC of PsA and RA compared to SFMC of OA.

synovial fluid and peripheral blood were stimulated with rIL-23, CD3/ D28 and PMA/Ionomycin as described earlier. Long term (6 days) stimulation of SFMCs of PsA patients with combination of CD3/CD28 and rIL-23 induced significantly more proliferation compared to shorter stimulation (4 h) with PMA/ionomycin (69.4 ± 2.06 vs 5.4 ± 1.15, p < 0.0001) or 6 days of CD3/CD28 alone (69.4 ± 2.06 vs. 12.83 ± 3.76, p < 0.0001) as assessed by CFSE dilution assay (Fig. 5). Similar results were observed with stimulated PBMCs of PsA patients. Combined CD3/CD28 and rIL-23 induced significantly more proliferation compared to CD3/CD28 alone (42.6 ± 1.43 vs. 9.72 ± 1.59, p < 0.0001) and PMA/ionomycin (42.6 ± 1.43 vs. 2.28 ± 1.8, p < 0.0001). These observations in SFMCs and PBMCs of PsA patients strongly indicate the mitogenic potential of rIL-23.

3.3. Functional significance of MAIT cells After characterizing the phenotype of MAIT cells in PsA patients, functional significance of these cells was determined by assessing IL17A production using flow cytometry. PBMCs and SFMCs were stimulated for 6 days with CD3, CD28, rIL-23, PMA, Ionomycin as described in methodology. On stimulation, synovial fluid MAIT cells of PsA patients produce significantly more IL-17A (32.66 ± 4.01%) compared to that of RA (23.93 ± 2.81%, p < 0.05) and OA (5.02 ± 0.157%, p < 0.0001). Similarly, peripheral blood MAIT cells of PsA (16.84 ± 1.83%, p < 0.01) and RA (12.34 ± 2.03%, p < 0.001) produced significantly more IL-17A than that of OA (5.02 ± 0.104%) on stimulation (Fig. 3). 3.4. Expression of IL-23R in MAIT cells

4. Discussion

As conventional IL-17A producing cells express IL-23R, we wanted to determine the expression of IL-23R on the IL-17A producing MAIT cells. In flow cytometry, upregulated IL-23R was noted in synovial fluid MAIT cells of PsA (24.97 ± 2.33%, p < 0.0001) and RA SFMC (21.93 ± 2.29%, p < 0.0001) compared to that of OA (2.13 ± 2.29) (Fig. 4A). Similar trends were noted in peripheral blood MAIT cells of PsA (18.21 ± 2.14%, p < 0.001), RA (16.58 ± 3.66%, p < 0.001) and OA patients (1.74 ± 0.13%). Further, the expression of IL-23R was substantiated by western blot with lysates of magnetically sorted Vα7.2TCR+CD3+ T cells using stimulated PBMCs. The expression of IL23R was normalized with α-tubulin. Stimulated magnetically sorted Vα7.2TCR+CD3+ T cells showed significant upregulation of IL-23R expression compared to unstimulated cells (3.5 + 0.301 vs 1, p < 0.001, Fig. 4B). This observation was further confirmed at gene expression by qPCR for IL-23R. IL-23R was significantly upregulated in stimulated Vα7.2TCR+CD3+ T cells compared to unstimulated T cells (2.814 + 0.331 vs 1, p < 0.001, Fig. 4C).

In several autoimmune diseases including psoriatic arthritis (PsA), IL-17A plays a crucial role in disease initiation, progression and maintenance and it has been shown that majority of IL-17A is produced by Th17 (CD4+) cells at the local disease site [6,8,9]. Mucosal associated invariant T (MAIT) cells are characterized by expression of CD3+Vα7.2TCR+CD161hi and are establishing its role in the pathogenesis of several autoimmune diseases. MAIT cells are predominantly of CD8+ subtype and have been shown to produce IL-17A [19,20,34]. Thus CD8+ IL-17A+ MAIT cells are unique and have led researcher to investigate this T cell subpopulation in several autoimmune diseases. In this study we delineated the functional role of MAIT cells in pathogenesis of PsA. In PsA patients, MAIT cells were found in lesser frequency in peripheral blood, whereas higher frequency of these cells was noted in paired synovial fluid suggesting their recruitment at the local disease site- joint (Fig. 1). Similar trend was noted in another inflammatory arthritis-RA, whereas in non-inflammatory arthritis (OA), MAIT cells were found more in peripheral blood compared to joint. This observation is in line with previous reports where it has been shown that frequency of MAIT cells were reduced in peripheral blood of ankylosing spondylitis, and inflammatory bowel disease (IBD) [23,24]. Majority of MAIT cells in synovial fluid of PsA patients were of memory

3.5. Functional significance of IL-23R expression in MAIT cells The functional significance of IL-23R in MAIT cells was determined by assessing mitogenic effect of IL-23 on MAIT cells. CD3+ T cells from 5

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Fig. 4. Expression of IL-23R on MAIT cells. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from psoriatic arthritis (PsA, n = 10), rheumatoid arthritis (RA, n = 10) and osteoarthritis (OA, n = 10) patients were studied for IL-23R expression by flow cytometry, western blot and qPCR. (A) A representative histogram of SFMC from PsA patients showing expression of IL-23R on MAIT cells (CD161hiVα7.2TCR+). Scatter plot showing significant upregulation of IL-23R in PBMC and SFMC of PsA and RA patients compared to OA patients. *p < 0.0001, PBMC of PsA and RA compared to PBMC of OA. **p < 0.001, SFMC of PsA and RA compared to SFMC of OA. In PsA and RA patients, more IL-23R expression was noted in SFMC compared to PBMC. The scatter plot depicts the average percentage of IL-23R+MAIT per patient, n = 10 per patient type. Each patient sample was done in triplicate. Results were expressed as Mean ± SEM. One-way ANOVA with Tukey multiple comparison test was used to determine statistical significance. (B) The IL-23R expression was determined by western blot with lysates of magnetically sorted Vα7.2TCR+CD3+ T cells using stimulated PBMCs of PsA patients and normalized with α-tubulin. A representative western blot showing upregulation of IL-23R expression on stimulated PBMCs of PsA patient. Bar diagram (n = 5) showing significant upregulation of IL-23R on stimulated PBMCs compared to unstimulated. (C) Bar diagram showing significant upregulation of IL-23R mRNA in stimulated PBMCs of PsA patients (n = 5) compared to unstimulated. All experiments were done in triplicate. Results were expressed as Mean ± SEM. One-way ANOVA with Tukey multiple comparison test was used to determine statistical significance.

Fig. 5. IL-23R is functionally active on MAIT cells. Functional significance of MAIT cells were determined by assessing mitogenic activity of these cells in presence of different stimulus (PMA/ionomycin, CD3/CD28, rIL-23) and mitogenic activity was assessed by CFSE dilution assay. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated as described in the methodology. (A) Representative FACS plots for proliferation of MAIT cells (CD3+CD161hiVα7.2TCR+) stimulated with either PMA/ionomycin or CD3/CD28 or rIL-23+CD3/CD28 at different time points. (B) Scatter plot (n = 10) showing significant more proliferation of SFMCs and PBMCs of PsA patients stimulated with rIL-23+CD3/CD28 compared to CD3/CD28 alone or PMA/ionomycin. Results were expressed as Mean ± SEM. One-way ANOVA with Tukey multiple comparison test was used to determine statistical significance. 6

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(CD45RO+) phenotype (Fig. 2), which suggests a potential for rapid clonal expansion at the local disease site and induction of inflammation. These MAIT cells in the synovial fluid and blood are functionally active as evidenced by upregulation of IL-17A production on stimulation (Fig. 3). Synovial fluid MAIT cells were functionally more active compared to that of blood. The maximum upregulation of IL-17A was noted in MAIT cells of PsA patients followed by RA, whereas minimal difference noted in OA. The conventional IL-17A producing cells (Th17) express IL-23R as IL-23 signaling is required to differentiate cells towards Th17 lineage. As MAIT cells are producing IL-17A, we sought to determine expression of IL-23R on these cells. Here we observed that synovial fluid and peripheral blood MAIT cells expressed IL-23R, and on stimulation upregulation of IL-23R was observed on these cells (Fig. 4). The expression of IL-23R was more in PsA and RA samples compared to OA. Further, this IL-23R is functionally active as evidenced my profound mitogenic effect on MAIT cells in presence of rIL-23 (Fig. 5). The mitogenic effect was evident on long term stimulation (6 days). This finding is pathologically more relevant as in diseased milieu T cells do get exposed to proinflammatory cytokines like IL-23 for a longer duration of time resulting in perpetuation of inflammation. This novel observation provides a new insight that MAIT cells are likely to be part of the IL-23/IL-17A cytokine network and critical for the pathogenesis of PsA. Of note, in a previous study our research group had shown enrichment of IL-17A producing CD4+ cells in the joints of PsA patients [6]. Though studies have shown that CD4+ Th17 cells are the major source of IL-17A in autoimmune diseases [4,6,8,35,36], but there are other cell populations which also secretes IL-17A at the local disease site. In this study our focus was to delineate the role of MAIT cells in the pathogenesis of psoriatic arthritis which are chiefly CD8+ T cells. Several evidence suggest potential pathogenic role of CD8+ T cells in psoriatic disease- (a) predominance of CD8+ T cells in psoriasis and PsA at disease site [4]; (b) MHC class I allele association in both psoriasis and PsA [37]; (c) IL-17A producing CD8+ T cells (Tc17) and innate MAIT cells found in abundance in psoriatic skin plaques [25]; (d) patients with advanced HIV (low CD4 count) develop psoriatic disease or worsening of existing psoriatic disease whereas RA gets better in advance HIV [38–40]; (e) disease severity of PsA positively correlates with frequency of IL-17A+CD8+ T cells in synovial fluid [26]. PsA is considered more similar to ankylosing spondylitis (AS) based on the immunopathogenesis [35]. In AS regulatory role of MAIT cells have been reported [24,41]. Inman and his research group [24] have demonstrated enrichment of IL-17A producing MAIT cells in the synovial fluid of AS patients whereas blood showed lower frequency of MAIT cells. In this study, we also have observed similar results in PsA. Our study demonstrated enrichment of Tc17 CD8+ /MAIT cells in the synovial fluid of PsA patients and majority of them are functionally active. This MAIT cells are of memory phenotype and express IL-23R. Identification of functionally active IL-23R on Tc17 CD8+ /MAIT cells in this study delineates a new dimension: (i) MAIT cells also belongs to the IL-23/IL-17 cytokine network; (ii) because of functional IL23R once these MAIT cells migrates to the joint synovium IL-23 can independently regulate these critical Tc17 CD8+ MAIT cells and may not need MR1 associated activation process.

Funding This project was supported by the VA Medical Center Sacramento. 7. Ethics approval and consent to participate IRB-VA Sacramento Medical Center. 8. Availability of data and material Data and materials are in possession with the authors (SPR). Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. Author contributions SKR designed experiments, supervised statistical analysis. CA performed all experiments with help from others and did the statistical analysis. AM has contributed in data analysis and in manuscript preparation. SPR conceived the study and helped in designing experiments. All authors have contributed to the conception and/or acquisition of data and analysis for this project and to either drafting or revising the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] D.D. Gladman, C. Antoni, P. Mease, D.O. Clegg, O. Nash, Psoriatic arthritis: Epidemiology, clinical features, course, and outcome, in: Ann. Rheum. Dis. 2005, 64 Suppl 2:ii14-17. [2] O. FitzGerald, R. Winchester, Psoriatic arthritis: From pathogenesis to therapy, Arthritis Res. Ther. 11 (1) (2009) 214. [3] P. Rahman, J.T. Elder, Genetic epidemioloqy of psoriasis and psoriatic arthritis, in: Ann. Rheum. Dis., 2005, 64 Suppl 2:ii37-39. [4] I. Kryczek, A.T. Bruce, J.E. Gudjonsson, A. Johnston, A. Aphale, L. Vatan, W. Szeliga, Y. Wang, Y. Liu, T.H. Welling, et al., Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis, J. Immunol. 181 (7) (2008) 4733–4741. [5] P.C. Res, G. Piskin, O.J. de Boer, C.M. van der Loos, P. Teeling, J.D. Bos, M.B. Teunissen, Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis, PLoS ONE 5 (11) (2010) e14108. [6] S.P. Raychaudhuri, S.K. Raychaudhuri, M.C. Genovese, IL-17 receptor and its functional significance in psoriatic arthritis, Mol. Cell. Biochem. 359 (1–2) (2012) 419–429. [7] A. Johnston, J.E. Gudjonsson, Psoriasis and the MAITing game: a role for IL-17A+ invariant TCR CD8+ T cells in psoriasis? J. Invest. Dermatol. 134 (12) (2014) 2864–2866. [8] S.P. Raychaudhuri, S.K. Raychaudhuri, Mechanistic rationales for targeting interleukin-17A in spondyloarthritis, Arthritis Res. Ther. 19 (1) (2017) 51. [9] E.A. Wang, E. Suzuki, E. Maverakis, I.E. Adamopoulos, Targeting IL-17 in psoriatic arthritis, Eur. J. Rheumatol. 4 (4) (2017) 272–277. [10] E. Toussirot, Ixekizumab: an anti- IL-17A monoclonal antibody for the treatment of psoriatic arthritis, Expert Opin. Biol. Ther. 18 (1) (2018) 101–107. [11] S.P. Raychaudhuri, A cutting edge overview: psoriatic disease, Clin. Rev. Allergy Immunol. 44 (2) (2013) 109–113. [12] R.P. Nair, K.C. Duffin, C. Helms, J. Ding, P.E. Stuart, D. Goldgar, J.E. Gudjonsson, Y. Li, T. Tejasvi, B.J. Feng, et al., Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways, Nat. Genet. 41 (2) (2009) 199–204. [13] R. Watanabe, A. Gehad, C. Yang, L.L. Scott, J.E. Teague, C. Schlapbach, C.P. Elco, V. Huang, T.R. Matos, T.S. Kupper, R.A. Clark, Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells, Sci. Transl. Med. 7 (279) (2015) 279ra39. [14] D. Hijnen, E.F. Knol, Y.Y. Gent, B. Giovannone, S.J. Beijn, T.S. Kupper, C.A. Bruijnzeel-Koomen, R.A. Clark, CD8(+) T cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of IFN-gamma, IL-13, IL17, and IL-22, J. Invest. Dermatol. 133 (4) (2013) 973–979. [15] V. Chandran, S.P. Raychaudhuri, Geoepidemiology and environmental factors of psoriasis and psoriatic arthritis, J. Autoimmun. 34 (3) (2010) J314–J321. [16] H. Valdimarsson, R.H. Thorleifsdottir, S.L. Sigurdardottir, J.E. Gudjonsson, A. Johnston, Psoriasis–as an autoimmune disease caused by molecular mimicry,

5. Conclusions 1. Synovial fluid MAIT cells were a major source of IL-17A and majority of MAIT cells were CD8+. Enrichment of these Tc17/MAIT cells in PsA SFMC and its association with MHC class-1 indicates that these cells could be of significant pathological significance. 2. Characterization of functionally active IL-23R on MAIT cells provides new dimensions: (i) MAIT cells plays a contributing role for the IL-23/ IL-17 cytokine network, (ii) IL-23 can regulate these critical Tc17 CD8+ MAIT cells independent of bacterial metabolites once these cells migrate to joint tissue. 7

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