Journal of Neuroimmunology 278 (2015) 239–246

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Association of circulating follicular helper T cells with disease course of NMO spectrum disorders Yu-Jing Li a, Fang Zhang a, Yuan Qi a, Guo-Qiang Chang a, Ying Fu a, Lei Su a, Yi Shen a, Na Sun a, Aimee Borazanci b, Chunsheng Yang a, Fu-Dong Shi a,b, Yaping Yan a,⁎ a b

Department of Neurology and Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin 300052, China Department of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA

a r t i c l e

i n f o

Article history: Received 2 July 2014 Received in revised form 5 October 2014 Accepted 9 November 2014 Keywords: NMOSD Tfh cells IL-21 IL-6

a b s t r a c t While follicular helper T (Tfh) cells have been shown to be involved in many autoimmune diseases, the association of Tfh cells with the disease activity of neuromyelitis optica spectrum disorders (NMOSDs) remains unclear. In this study, the CD4+ CXCR5+ PD-1+ Tfh cell population in peripheral blood mononuclear cells (PBMCs) obtained from NMOSD patients, age- and gender-matched healthy controls, and multiple sclerosis patients was compared by flow cytometry. The serum levels of IL-21, IL-6, IL-17, TNF-α and IL-10 were analyzed by ELISA assays. We found that in NMOSD, the Tfh cell frequency is higher than that of healthy subjects and multiple sclerosis (MS) patients. There are more Tfh cells in the relapsing stage than the remitting stage of NMOSD, thus demonstrating the close association of the Tfh cell population with disease activity. Methylprednisolone, which is used to control disease relapses, significantly decreased the proportion of Tfh cells in NMOSD patients. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Neuromyelitis optica (NMO) is a severe inflammatory demyelinating disorder of the central nervous system (CNS), which is clinically characterized by severe attacks of myelitis and optic neuritis (ON) (Jarius et al., 2008). Limited forms of the disease are referred to as NMO spectrum disorders (NMOSDs) and include patients with either ON or longitudinally extensive transverse myelitis (LETM) (single or recurrent events of LETM or recurrent or simultaneous bilateral ON) (Wingerchuk et al., 2007). The presence of a circulating autoantibody, termed NMO-IgG, directed against the extracellular domain of the water channel protein aquaporin-4 (AQP4) expressed in astrocytes, is a characteristic feature of NMO that distinguishes NMO from other inflammatory demyelinating diseases (Lennon et al., 2005). Around 90% of NMO and more than 50% of NMOSD patients are NMO-IgG seropositive (Waters et al., 2012). Accumulating evidence has shown that NMO-IgG is pathogenic and often correlates with disease severity (Jarius and Wildemann, 2010). It is thought that the B cell or plasma cell-produced NMO-IgG binding to astrocytic AQP4 activates complement deposition (Lucchinetti et al., 2002; Misu et al., 2007; Saadoun et al., 2010), leading to astrocyte damage and inflammatory reaction

Abbreviations: NMOSD, neuromyelitis optica spectrum disorder; Tfh, follicular helper T; NMO, neuromyelitis optica; MS, multiple sclerosis; HC, health control. ⁎ Corresponding author at: Department of Neurology and Tianjin Neurological Institute Tianjin Medical University General Hospital, No. 154 Anshan Road, Tianjin 300052, China. Tel./fax: +86 22 60817429. E-mail address: [email protected] (Y. Yan).

http://dx.doi.org/10.1016/j.jneuroim.2014.11.011 0165-5728/© 2014 Elsevier B.V. All rights reserved.

(Kinoshita et al., 2009) with leukocyte infiltration (Saadoun, Waters, 2010) and cytokine release (Uzawa et al., 2010), thus resulting in disease development. Even in NMO seronegative patients, the presence of antibodies against myelin antigens has been reported (Kitley et al., 2014; Kitley et al., 2012; Rostasy et al., 2013; Sato et al., 2014). Although the role of these antibodies has not been clarified, it is anticipated that humoral autoimmunity would also play a role in immune pathology in these patients. Follicular helper T (Tfh) cells are antigen experienced CD4+ T cells found in B cell follicles of secondary lymphoid organs and are identified by their constitutive expression of B cell follicle homing receptor CXC chemokine receptor 5 (Breitfeld et al., 2000, Fazilleau et al., 2009). The main function of Tfh cells is to help B cell activation and antibody production in humoral immune responses, specifically through interactions between molecules on the surface of Tfh cells and receptors or ligands on the surface of B cells (Morita et al., 2011). IL-21 is a well-known pro-inflammatory cytokine which is preferentially expressed by Tfh cells and is an important regulator of humoral responses by directly regulating B cell proliferation, maturation, and class switching (Kuchen et al., 2007; Spolski and Leonard, 2008). In addition, IL-6 and IL-21 are critical cytokines for Tfh cell differentiation (Nurieva et al., 2008). It has been reported that dysregulated Tfh cells can cause systemic autoimmunity and autoantibody production or contribute to T cellmediated organ-specific autoimmunity (Vinuesa et al., 2005). Zhang et al. has shown that in comparison with healthy controls, a higher frequency of CD4+ CXCR5+ Tfh cells was found in newly diagnosed IgA nephropathy (Zhang et al., 2014a). Recently, it was found that thymic Tfh cells are also involved in the pathogenesis of myasthenia

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Table 1 Demographic and clinical characteristic of patients with NMOSD, MS and HC. Characteristics

NMOSD

MS

HC

Pa

n Age (year) Female, n (%) Duration of disease (year) ARR No of attack EDSS AQP4-Ab positive n (%) NMO/NMOSD-ON/NMOSD-LETM

35 46.54 ± 13.07 (20–68) 30 (85.7) 7.00 ± 7.40 (1–34) 0.94 ± 0.5 (0.20–2) 4.77 ± 3.87 (1–17) 4.53 ± 2.51 (1–9) 25 (71.4) 30/2/3

20 39.85 ± 12.14 (21–61) 13 (65) 4.95 ± 6.23 (0.5–26) 1.02 ± 0.82 (0.15–) 3.00 ± 2.41 (1–9) 2.50 ± 1.42 (1–6) 0 (0) –

20 48.40 ± 14.28 (22–68) 18 (90) – – – – – –

0.096 0.084 0.142 0.893 0.070 0.001 b0.0001 –

Data are expressed as Mean ± SD (range) unless otherwise stated. NMOSD = Neuromyelitis optica (NMO) spectrum disorders; MS = multiple sclerosis; HC = health control; EDSS = Expanded Disability Status Scale; ON = optic neuritis; LETM = longitudinally extensive transverse myelitis. AQP4-Ab = aquaporin-4 antibody; ARR = annual relapse rate. a Refers to p values obtained following the comparison among the MS, NMOSD and HC by means of an ANOVA test (age, duration, EDSS, number of attacks, ARR) and chi-square test (gender, AQP4 positive).

gravis with thymoma (Zhang et al., 2014b). Ma et al. reported that the Tfh cell population and serum IL-21 concentration were significantly increased in rheumatoid arthritis patients (Ma et al., 2012). It was summarized that the Tfh cell frequency was abnormal in several autoimmune diseases and their animal models, especially in those humoral immunity-mediated autoimmune diseases (Yu and Vinuesa, 2010). However, little is known about the relationship of Tfh cells with NMOSD. In this study, we quantified the frequency of Tfh and attempted to correlate it with disease activity of NMOSD. 2. Materials and methods 2.1. Participants Informed consent was obtained from all participants and the study was approved by the Tianjin Medical University General Hospital Institutional Review Board and Ethics Committee. The recruitment of this study was based on a clinical database beginning from January 2013 to March 2014. On the basis of medical records, disease duration and annual relapse rates were recorded, as well as the disease-modifying and symptomatic medications that were used. Serum AQP4-IgG was measured with FACS-based assay. On the basis of interviews and neurological examinations, the current degree of each patient's disability was assessed in the Expanded Disability Status Scale (EDSS). For the MRI evaluation, 3 Tesla GE scanners were used at admission with a comprehensive MRI protocol, including T1, T2, FLAIR and contrast-enhanced T1-weighted imaging (CET1-WI) of the brain and spine. The patients were enrolled based on the criteria as follows: (1) The NMOSD patients who met the diagnostic criteria proposed by Wingerchuk et al. in 2007 Table 2 Demographic and clinical characteristic of patients with relapsing and remitting NMOSD. Characteristics

Relapsing

Remitting

N Age (year)

11 48.18 ± 11.92 (22–64) 10 (90.9) 6.91 ± 5.96 (1–20) 1 ± 0.54 (0.31–2) 5.55 ± 3.98 (1–14) 4.82 ± 2.39 (2–8) 7 (63.6) 9/1/1

24 45.79 ± 13.74 (20–68) 20 (83.3) 7.04 ± 8.10 (1–34) 0.89 ± 0.48 (0.20–2) 4.42 ± 3.84 (1–17) 4.40 ± 2.60 (1–9) 18 (75) 21/1/2

Female, n (%) Duration of disease (year) ARR No of attack EDSS AQP4-positive n (%) NMO/NMOSD-ON/NMOSDLETM

Pa 0.657 1.000 0.747 0.386 0.370 0.484 0.689 –

Data are expressed as mean ± SD (range) unless otherwise stated. NMOSD = Neuromyelitis optica (NMO) spectrum disorders; MS = multiple sclerosis; EDSS = Expanded Disability Status Scale; ON = optic neuritis; LETM = longitudinally extensive transverse myelitis. AQP4 = aquaporin 4; ARR = annual relapse rate. a Refers to p values obtained following the comparison between relapsing NMOSD and remitting NMOSD by means of a Mann–Whitney U test(age, duration, EDSS, number of attacks, ARR) and chi square test (gender, AQP4 positive).

(Wingerchuk et al., 2007); (2) the MS patients who met the McDonald Criteria of MS as revised in 2010 (Polman et al., 2011); (3) all patients had no corticosteroid or other immunosuppressant therapy in the last 8 weeks; and (4) all patients had no other autoimmune diseases. We prospectively screened 302 NMOSD and MS patients from a neurology department that specialized in inflammatory diseases of the central nervous system. Fifty-five patients who met the criteria were enrolled: this included 35 patients with NMOSD (24 remitting patients and 11 relapsing patients), and 20 patients with MS (11 remitting patients and 9 relapsing patients). In addition, 20 age- and gender-matched healthy controls (HCs) were included in this study, and none of them had a history of disease or infection during the previous 8 weeks. For the detection of IL-17, IL-10 and TNF-α, the serum samples from 20 NMOSD patients (10 remitting and 10 relapsing), 10 MS patients, and 10 age- and gender-matched healthy controls (HCs) were included. 2.2. Treatment and follow-up To elucidate the dynamic changes of Tfh in different disease stages, we followed up on 11 relapsing patients (no corticosteroid and other immunosuppressant therapy in the last 8 weeks since this attack). Five relapsing patients were excluded due to a urinary tract infection (n = 4) and a suspected lung infection (n = 1) in the hospital. Patients with new neurological symptoms lasting at least 24 h and accompanied by new neurological examination findings and/or new lesions on MRI were defined as relapse. The clinical remission was defined as both neurological symptoms and neurological examination signs that remained stable for at least 30 days from last relapse. All six relapsing patients were given 500 mg/day methylprednisolone (MPDN) venous transfusion for the first 3 days and gradually decreased to maintenance dosage prednisolone (PDN) which was orally administered. All six patients showed effective response to the PDN treatment. Venous blood samples were collected before MPDN treatment and at least four weeks after treatment. 2.3. PBMC isolation and flow cytometry All the blood samples were collected into tubes containing EDTA, and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using a lymphocyte-separating medium (Mediatech, Manassas, VA). The cells were washed twice with PBS, resuspended with the frozen stock solution (FBS containing 10% DMSO) and stored in liquid nitrogen through the gradient freezing process. For cell staining, as the CD4+ CXCR5+ ICOS+ or CD4+ CXCR5+ PD-1+ was observed in several autoimmune diseases(Breitfeld et al., 2000; Fazilleau et al., 2009; Kuchen et al., 2007; Morita et al., 2011; Uzawa et al., 2010), we chose the CD4+ CXCR5+ PD-1+ as the marker for Tfh. The frozen cells were recovered in 37 °C immediately after taken from liquid nitrogen, washed once and re-suspended with staining buffer,

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Table 3 Demographic and clinical characteristics of six followed up NMOSD patients. Patients

Diagnose

Age (year)

Gender

Disease duration (year)

Number of attacks (year)

ARR

EDSS

AQP4-Ab

Patient1 Patient2 Patient3 Patient4 Patient5 Patient6

NMO NMO NMO NMO NMO LETM

64 41 51 43 40 60

F F F F F F

4 1 9 13 20 1

4 1 7 4 9 2

1.00 1.00 0.78 0.31 0.45 2.00

8.5 8.5 3 2.5 3.5 6

Positive Positive Positive Negative Positive Positive

NMOSD = neuromyelitis optica (NMO) spectrum disorders; EDSS = Expanded Disability Status Scale; LETM = longitudinally extensive transverse myelitis. AQP4 = aquaporin 4; ARR = annual relapse rate; F = female.

then incubated at room temperature with CD4-Percp/Cy5.5, CXCR5FITC, PD-1-PE mouse anti-human mAbs (BioLegend, San Diego, CA) for 45 min. After washing, the cells were then re-suspended in 400 μL of staining buffer and analyzed on FACSCalibur or FACSAriaIII (BD Biosciences, Franklin lakes, NJ). The data was then analyzed using FlowJo 7.6.

in all patients' serum. Fixed cells were sequentially incubated with patients' serum and Percp–Cy5.5-conjugated goat anti-human IgG Antibody (BioLegend, America). Cells were analyzed on Calibur (BD Biosciences), and Percp-mean fluorescence intensity (MFI) was measured on EGFP positive cells. The EGFP only transfect HEK-293T cell line was used as control. A serum was considered AQP4-IgG positive if it bounds to AQP4-EGFP-HEK-293T cells but not EGFP-HEK-293T cells.

2.4. Serum cytokine level detection 2.6. Concentration of AQP4-antibody detection Serum IL-21, IL-6, IL-17, IL-10 and TNF-α levels were measured using enzyme-linked immunosorbent assay (ELISA) (BioLegend, San Diego, CA for IL-21, and R&D System, Minneapolis, MN for IL-6, IL-17, IL-10 and TNF-α) according to the manufacturer's instructions. Optical densities were measured at 450 nm and 570 nm.

Serum AQP4-antibody level was measured using enzyme-linked immunosorbent assay (ELISA) (Yuanye Bio-Technology Co., Shanghai, China) according to the manufacturer's instructions. Optical densities were measured at 450 nm.

2.5. FACS-based AQP4-IgG detection

2.7. Statistical analysis

An AQP4 (M1)-EGFP fusion gene transfected HEK-293T cell line was used as the antibody harboring cell line to detect the AQP4-IgG

Statistical analysis was performed using GraphPad Prism version 4. Continuous variables were reported as means ± SD and categorical

Fig. 1. The comparison of Tfh cells among the NMOSD/MS/HC groups. (A) The strategy for gating the CXCR5 and PD-1 double positive cells from CD4+ cells. High-expressing CD4 cells were first gated from the Peripheral Blood Mononuclear cells. Numbers above the outlined area indicates the average frequency of CD4 positive cells in HC/NMOSD/MS group. The CXCR5 and PD-1 double positive cells were then gated from the CD4+ population. The number above the outlined area was the average frequency of Tfh in HC/NMOSD/MS groups (B) Comparison of CD4+ T cells (C) Comparisons of Tfh cell. Each data points represent an individual subject. The horizontal lines show the mean ± SD in each group. The data are representative of three independent experiments.

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Fig. 2. Serum concentrations of cytokines in HC/NMOSD/HC. (A) Comparison of serum IL-21 level. (B) Comparison of serum IL-6 level. (C) Comparison of serum TNF-α level. (D) Comparison of serum IL-17 level. (E) Comparison of serum IL-10 level. Each data points represents an individual subject. The horizontal lines show the mean ± SD in each group. The data are representative of two independent experiments.

variables appeared as percentages. Comparison among the MS, NMOSD and HC by means of one-way ANOVA test (age), Mann–Whitney test (duration, EDSS, number of attacks, ARR) and chi-square test (gender, AQP4 positivity). We applied student's t test for quantitative data and chi-squared test or Fisher exact test for qualitative data. For the six follow-up patients, paired t test was applied to compare the data in relapse and remission stage. One-tailed spearman test was used to analyze the correlations between the AQP4-antibody level and Tfh cell count. A p-value of ≤0.05 was considered statistically significant.

3. Result 3.1. Patients' characteristics No significant difference was found in these three groups in age and proportion of females. The disease duration of NMOSD patients and MS patients showed no significant difference. There was also no significant difference in annual relapse rate (ARR) and number of attacks. There was significant difference between the MS and NMOSD groups in

Fig. 3. The comparison of Tfh cells and serum cytokine levels between the relapsing and remitting NMOSD and MS. (A) Comparison of frequency of CXCR5+PD-1+ cells in CD4+ T cells among 20 Health Control, 11 Relapsing NMOSD, 24 Remitting NMOSD; (B) Comparison of frequency of CXCR5+PD-1+cells in CD4+ T cells among 20 Health Control, 9 Relapsing MS, 11 Remitting MS; (C) Comparison of serum IL-21 level among HC, relapsing and remitting NMOSD; (D) Comparison of serum IL-6 level among HC, relapsing and remitting NMOSD; (E) Comparison of serum TNF-α level among HC, relapsing and remitting NMOSD; (F) Comparison of serum IL-17 level among HC, relapsing and remitting NMOSD; (G) Comparison of serum IL-10 level among HC, relapsing and remitting NMOSD. During IL-21 and IL-6 comparison, 20 Health Control, 11 Relapsing NMOSD, 24 Remitting NMOSD patients were involved. When comparing the TNF-α, IL-17 and IL-10, 10 Health Control, 10 Relapsing NMOSD and 10 Remitting NMOSD patients were involved. Each data points represent an individual subject. The horizontal lines show the mean ± SD in each group. The data are representative of three independent experiments.

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Fig. 4. The comparison of Tfh cells and serum cytokine levels in six followed-up NMOSD patients (A) Comparison of frequency of CXCR5+PD-1+ cells. (B) Comparison of serum IL-21 level. (C) Comparison of serum IL-6 level. (D) Comparison of serum TNF-α level. (E) Comparison of serum IL-17 level. (F) Comparison of serum IL-10 level. Each line represents the changes of Tfh and levels of cytokines in relapsing (before treatment) and remitting (after treatment) stage of these six patients. The data are representative of three independent experiments.

EDSS and AQP4-Ab positivity rate. The 35 patients with NMOSD include: 30 patients with definite NMO and 5 patients with limited form including 2 patients with recurrent bilateral ON and 3 patients with single or recurrent events of LETM. The patients for the comparison of remitting and relapsing NMOSD and the six follow-up patients were also from these three groups. The demographic and clinical characteristics were shown respectively in Tables 1–3. 3.2. Augmented circulating Tfh cells in NMOSD patients To elucidate the potential differences of CD4+ CXCR5+ PD-1+ Tfh cells in the NMOSD/healthy control (HC)/MS group, the fresh-frozen PBMCs from all the participants were stained by fluorescence-labeled anti-CD4/CXCR5/PD-1 mouse anti human monoclonal antibodies and analyzed by flow cytometry. The results indicated that there was no significant difference in the percentage of circulating CD4+ T cells between NMOSD and HC. However, the CD4+ T cell population was higher in MS than in NMOSD and HC (Fig. 1A and B). Interestingly, among the CD4+ T cells, the CXCR5 and PD-1 double positive Tfh population were

significantly higher in NMOSD than in HC and MS. There was no difference in the Tfh cell population in CD4+ T cells between MS and HC (Fig. 1C). 3.3. Increased serum IL-21 and IL-6 levels in NMOSD patients IL-21 and IL-6 played important roles in Tfh cell differentiation (Lennon et al., 2005) and it was shown that IL-21 was mainly produced by Tfh cells. The serum IL-21 and serum IL-6 were determined by ELISA assays. As shown in Fig. 2A, similarly to the Tfh cell populations, the IL-21 level in NMOSD was significantly higher than HC and MS. The serum IL-6 level was also significantly higher in NMOSD than HC and MS (Fig. 2B). The serum concentration of IL-21 in MS was higher than HC, but there was no difference in serum IL-6 between these two groups (Fig. 2A, B). This data indicated a higher frequency of CXCR5+ PD-1+ CD4+ Tfh cells and significantly more elevated levels of closely related serum cytokines in patients with NMOSD than in HC and MS patients. Comparatively, serum concentrations of other inflammatory and anti-inflammatory cytokines including TNF-α, IL-17 and IL-10 were also compared between groups. Serum from 20 NMOSD patients (10 remitting and 10 relapsing patients), 10 MS patient and 10 age- and gender-matched healthy control (HC), were randomly chosen to detect the concentration of TNF-α, IL-17 and IL-10 by ELISA. As shown in Fig. 2C, D and E, IL-17, IL-10 and TNF-α levels turned out to be significantly higher in both NMOSD and MS patients compared to HC, but no significantly difference was found between NMOSD and MS groups. Interestingly, we found a trend of higher IL-17 and lower IL-10 in NMOSD compared to MS patients (Fig. 2D and E). 3.4. The frequency of blood Tfh cells correlated with disease activity in NMOSD patients

Fig. 5. Correlation between AQP4-antibody concentration and Tfh cell counts. Each data point represents an individual subject.

To find the association of circulating Tfh cells with disease activity, Tfh cell frequency was compared between relapsing and remitting NMOSD patients. As shown in Fig. 3A, among the CD4+ T cells, the CXCR5 and PD-1 double positive Tfh population were significant higher in relapsing than in remitting patients. When compared to the HC group, the Tfh cell population was significantly higher even in remitting

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stage of NMOSD (Fig. 3A). On the contrary, there was no difference of the CXCR5 and PD-1 double positive Tfh population between relapsing MS patients, remitting MS and health subjects (Fig. 3B). Consistent with differences of Tfh cell population among all the comparisons, the serum level of IL-21 and other inflammatory cytokines including IL-6, TNF-α and IL-17 in relapsing NMOSD was significantly higher than in remitting NMOSD and HC (Fig. 3C–F). However, no significant difference was found in serum IL-10 level between relapsing and remitting NMOSD patients (Fig. 3G). Additionally, the serum concentration of IL-21 and IL-17 was higher in remitting NMOSD than HC. The above data indicated a higher frequency of Tfh cells and significantly elevated levels of closely related serum cytokines in patients with relapsing than remitting NMOSD. 3.5. Methylprednisolone treatment decreased the proportion of Tfh cells in NMOSD patients The circulating Tfh cell populations and serum IL-21, IL-6, TNF-α, IL-17 and IL-10 levels were monitored among the regular methylprednisolone treatment, which was the most preferred treatment to control disease relapse in our center. We followed up six NMOSD patients who received the methylprednisolone treatment and had an effective response. The results showed that the Tfh cell population was significantly decreased after treatment (Fig. 4A). Similar changes of serum IL-21, IL-6, TNF-α and IL-17 levels were also found after treatment (Fig. 4B–E), while no significant changes were found in IL-10 after treatment (Fig. 4F). Thus, the methylprednisolone treatment significantly inhibited the Tfh response and decreased the levels of serum inflammatory cytokines. 3.6. No correlations between AQP4-antibody level and Tfh cell counts We collected blood samples from AQP4-Ab positive NMOSD patients, and the lymphocytes were isolated by lymphocyte isolation buffer and were counted. The CD4+ CXCR5+ PD-1+ Tfh cell population was analyzed by flow cytometry. The Tfh cell counts per miniliter blood were calculated according to lymphocyte numbers and Tfh cell frequency in lymphocytes. The titers of AQP4-Ab in these patients were detected by ELISA. We then analyzed the correlation between the anti-AQP4 autoantibody titers and the Tfh cell counts. No correlation was found between AQP4-antibody titers and Tfh cell count in AQP4-Ab positive NMOSD patients (Fig. 5). 4. Discussion We have found a close association of Tfh cells with the disease activity of NMOSD, but how the Tfh cells favor disease pathogenesis still remains a question to be uncovered in the future. Tfh cells were first described more than 10 years ago as CD4+ T cells that reside in B cell areas of secondary lymphoid tissues in humans (Breitfeld et al., 2000). The main function of Tfh cells is to support B cell activation, expansion, and differentiation (Ansel et al., 1999; Hardtke et al., 2005; Tackenberg et al., 2007). Due to their function in mediating humoral immune responses, Tfh cells are thought to be involved in immunodeficiency when under-activated or autoimmunity when over-activated (Luo et al., 2013; Tackenberg et al., 2007). However, traditional Tfh cells were found to be located in secondary lymphoid tissues, which hindered the study of these cells in human diseases, as access to these tissues is often not feasible. The identification of circulating counterparts to tissue Tfh cells and the identification of Tfh cells in the peripheral blood have been instrumental to the study of these cells in human diseases. Recently, numerous findings have shown that aberrant Tfh cell activity is involved in immunopathology, especially in autoimmune diseases. It was shown that circulating Tfh cells were expanded in myasthenia gravis (MG), and Tfh cells were associated with disease

scores (Luo et al., 2013; Zhu et al., 2012). It was also reported that IL-21 was increased in PBMCs from MG patients (Zhu et al., 2012). Martin et al. (2012) reported that the levels of circulating Tfh cells were increased in autoimmune thyroid diseases and CD4+ T cells from patients with Graves' disease or Hashimoto's thyroiditis displayed increased IL-21 secretion and expression following in vitro culture. In juvenile dermatomyositis, an increase of CXCR5+ CCR6+ Th17-like and CXCR5+ CXCR3− CCR6− Th2-like Tfh cells was detected, and the disease score (Luo et al., 2013; Zhu et al., 2012) and the plasmablast numbers correlated positively with these Tfh cells (Wong et al., 2010). An increase in Tfh cells and serum IL-21 levels was also observed in systemic lupus erythematosus (Feng et al., 2012; Li et al., 2012), and Sjogren's syndrome patients (Fan et al., 2012). Numerous groups have investigated the role of Tfh cells in rheumatoid arthritis (RA), and found an increase in circulating Tfh cells and IL-21 level in RA which suggested the involvement of Tfh cells in the disease-process of RA (Ma et al., 2012; Rasmussen et al., 2010, Romme Christensen et al., 2013, Wang et al., 2013a). There was another report that peripheral ICOS+ CXCR5+ Tfh cells were increased in multiple sclerosis (MS) patients (Tangye et al., 2013). The aberrant production of high-affinity cross-reactive autoantibodies by the Tfh cell is very important in many autoimmune diseases (Ratelade and Verkman, 2012) including NMO. Although the definite pathogenesis is not clear, AQP4-Ab based antibody-dependent cellmediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) process in the pathogenesis has been reported as consistent with the pathological lesions in NMO patients (Ma and Deenick, 2014; Misu et al., 2013). Here, we found Tfh cells increased in NMOSD patients and Tfh cell population was associated with disease activity. The findings hold the potential of Tfh cells or related cytokines to be a biomarker of disease. In our research, we also compared the Tfh cell population between MS and HC and found that there was no difference between the CXCR5+ PD1+ Tfh cells in CD4+ T cells, which did not support the previous report on Tfh in the MS study. The most likely reason for this difference was due to the fact that the two groups analyzed different subsets of Tfh cells. Here we analyzed CXCR5+ PD1+ Tfh cells while the Christensen group reported ICOS+ CXCR5+ Tfh cells. Now, scientists realize that Tfh cells are heterogeneous as there are different subsets of Tfh cells that have been studied, including CXCR5+ ICOS+, CXCR5+ ICOShi, CXCR5+ PD1+, CXCR5+ PD1hi, CXCR5+ ICOS+ PD1+, CXCR5+ CD57+ and CXCR5+ IL-21+ (Polman et al., 2011). Since different groups use different markers for Tfh cell identification, further investigation is warranted to determine whether these markers are defining a different population of Tfh cells or the same population that expresses all the different markers. Corticosteroid was reported to inhibit the Tfh cell response by reducing the serum glucocorticoid-regulated kinase 1(SGK1). This was closely related to the survival of the cells through the down-regulating of IL-6 and IL-21 in SLE and IgA nephropathy patients (Zhang et al., 2014a). To identify the Tfh cell response to corticosteroid treatment in NMOSD patients, we followed up six patients showing effective response to PDN treatments. The significant decrease in frequency of Tfh cells and IL-21/IL-6 levels after treatment expanded our understanding that the Tfh population was partly involved in the disease relapsing course of NMOSD. IL-17, IL-10 and TNF-α have been reported to be closely related to inflammatory demyelination diseases in CNS. Wang et al. reported that Th17 cells were activated and serum IL-17 was elevated in both NMO and MS patients (Wang et al., 2011). Consistent with the reports, our study also showed that both NMOSD and MS patients have higher level of IL-17. Though IL-10 was commonly reported as an antiinflammatory cytokine, our results supported a previous report that a higher level of IL-10 was found in NMO than HC (Tangye et al., 2013; Wang et al., 2013b). Since TNF-α was a pro-inflammatory cytokine, more studies focused on TNF-α in MS. Several studies have revealed an elevated TNF-α level in autopsies, cerebrospinal fluid (CSF) and serum of MS patients (Alatab et al., 2011; Benvenuto et al., 1991;

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Martins et al., 2011; Rohowsky-Kochan et al., 2000; Trenova et al., 2011). Fewer studies pay attention to TNF-α in NMOSD patients. Wang et al. (Wang et al., 2013b, Wang et al., 2012) reported that no significant difference was found between MS and NMOSD patients, but they were significantly higher than HC, which was similar to our results. Although AQP4-Ab has satisfactory sensitivity and specificity for NMOSD diagnosis and it was also considered as one of the important pathogenic factor in NMOSD development, it was known that the titer of AQP4-Ab was not correlated with disease activity (Chanson et al., 2013; Hinson et al., 2009). Here, we also found no correlation between AQP4-Ab titer and Tfh cell counts. Though we found the association of Tfh cells with NMOSD, further study is needed to determine how Tfh cells are involved in NMOSD pathogenesis. In conclusion, through the findings of this study, we were able to describe the association of Tfh cells with NMOSD disease activity for the first time. As mentioned above, Tfh cells were heterogeneous. In NMOSD, a comprehensive study is needed to reveal the level of heterogeneity within the CXCR5+ population and Tfh cells, as different criteria in NMOSD need to be further compared. The association of different marker-expressed Tfh cells with disease activity of NMOSD needs to be explored in the future. It is also important to uncover the function of different Tfh cells in disease development in the future. Only when the questions mentioned above are answered, can we then effectively identify the role of Tfh cells in NMOSD. Overall, our current findings suggest that the involvement of Tfh cells in NMOSD should provoke more studies on Tfh cells as a potential pathogenic element and therapeutic target in this disease. Role of the sponsors The funding agencies had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review or approval of the manuscript or in the decision to submit the manuscript for publication. Conflict of interest statement The authors do not have a commercial or other association that might pose a conflict of interest. Acknowledgments We would like to acknowledge Chinese Natural Science Foundation (81100888, 81371372) and the National Key Clinical Specialty Construction Program of China for funding this research. We thank our patients for participating in this study; as well as Ms J. Zhou, C. Wang and the neuroimmunology laboratory team for their technical support. References Alatab, S., Maghbooli, Z., Hossein-Nezhad, A., Khosrofar, M., Mokhtari, F., 2011. Cytokine profile, Foxp3 and nuclear factor-kB ligand levels in multiple sclerosis subtypes. Minerva Med. 102, 461–468. Ansel, K.M., McHeyzer-Williams, L.J., Ngo, V.N., McHeyzer-Williams, M.G., Cyster, J.G., 1999. In vivo-activated CD4 T cells upregulate CXC chemokine receptor 5 and reprogram their response to lymphoid chemokines. J. Exp. Med. 190, 1123–1134. Benvenuto, R., Paroli, M., Buttinelli, C., Franco, A., Barnaba, V., Fieschi, C., et al., 1991. Tumour necrosis factor-alpha synthesis by cerebrospinal-fluid-derived T cell clones from patients with multiple sclerosis. Clin. Exp. Immunol. 84, 97–102. Breitfeld, D., Ohl, L., Kremmer, E., Ellwart, J., Sallusto, F., Lipp, M., et al., 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. 192, 1545–1552. Chanson, J.B., Alame, M., Collongues, N., Blanc, F., Fleury, M., Rudolf, G., et al., 2013. Evaluation of clinical interest of anti-aquaporin-4 autoantibody followup in neuromyelitis optica. Clin. Dev. Immunol. 2013, 146219. Fan, L., Wang, Q., Liu, R., Zong, M., He, D., Zhang, H., et al., 2012. Citrullinated fibronectin inhibits apoptosis and promotes the secretion of pro-inflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis. Arthritis Res.Ther. 14, R266. Fazilleau, N., Mark, L., McHeyzer-Williams, L.J., McHeyzer-Williams, M.G., 2009. Follicular helper T cells: lineage and location. Immunity 30, 324–335.

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