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Exploring the imbalance of circulating follicular helper CD4+ T cells in sarcoidosis patients

Open AccessPublished:February 08, 2020DOI:https://doi.org/10.1016/j.jdermsci.2020.02.002

      Highlights

      • Circulating follicular helper CD4+ T (TFH) cell subsets are imbalanced in sarcoidosis.
      • TFH cells may migrate to the affected tissues.
      • Circulating TFH are one of the potential cell types capable of producing IL-17.
      • Circulating TFH help to enhance Th17 responses, and may promote the chronic inflammation.
      • We could not demonstrate a direct linkage between the imbalance of TFH cells and B cell alteration in sarcoidosis.

      Abstract

      Background

      Sarcoidosis is a systemic granulomatous disease characterized by the combination of Th1 and Th17 responses. Recently, several arguments have suggested a potential involvement of B cells as well as T cells in the pathogenesis of sarcoidosis. Follicular helper CD4+ T (TFH) cells are specialized in interacting with and helping B cells, and play a crucial role in the formation of germinal centers.

      Objective

      We sought to explore the status of TFH cells and investigate their possible pathogenic role in sarcoidosis.

      Methods

      TFH cells and B cells in peripheral blood were examined by flow cytometry, and serum samples were studied by cytokine arrays. Immunohistochemistry was performed to check for the presence of TFH cells in sarcoidosis skin lesions. Gene expression in isolated TFH cells was analyzed by quantitative RT-PCR.

      Results

      The proportion of circulating TFH cells was decreased. CD4+CXCR5+ TFH cells were observed in cutaneous lesions in sarcoidosis. Gene expression in circulating TFH cells and serum cytokine concentrations related to Th17 were increased in sarcoidosis patients. Gene expressions of B cell differentiation cytokines in TFH cells were not altered in sarcoidosis patients.

      Conclusion

      We herein describe a decrease of circulating TFH cells and their migration to affected tissues. Circulating TFH cells are one of the potential cell types capable of producing IL-17 and enhancing Th17 responses, and may promote the chronic inflammation. We could not demonstrate a direct linkage between the imbalance of TFH cells and abnormal B cell differentiation in sarcoidosis.

      Keywords

      1. Introduction

      Sarcoidosis is a systemic disease of unknown etiology characterized by the formation of granulomas in various organs [
      • Valeyre D.
      • et al.
      Sarcoidosis.
      ]. T helper type 1 (Th1) responses and the Th1-associated cytokines have crucial roles in the pathogenesis of sarcoidosis. Recently, increased Th17 production and interleukin-17 (IL-17) positive cellular infiltration were reported [
      • Ten Berge B.
      • et al.
      Increased IL-17A expression in granulomas and in circulating memory T cells in sarcoidosis.
      ]. Although the role of Th17 cells in the pathogenesis of sarcoidosis remains unclear, Th17 has been demonstrated to be important for promoting chronic inflammation in sarcoidosis and various autoimmune diseases [
      • Ten Berge B.
      • et al.
      Increased IL-17A expression in granulomas and in circulating memory T cells in sarcoidosis.
      ,
      • Facco M.
      • et al.
      Sarcoidosis is a Th1/Th17 multisystem disorder.
      ].
      Furthermore, some recent studies suggested the potential involvement of B cells in the pathogenesis of this disease; B cell depletion using anti-CD20 antibody has been used as an effective therapy for sarcoidosis [
      • Saussine A.
      • et al.
      Active chronic sarcoidosis is characterized by increased transitional blood B cells, increased IL-10-producing regulatory B cells and high BAFF levels.
      ,
      • Cinetto F.
      • et al.
      Rituximab in refractory sarcoidosis: a single centre experience.
      ,
      • Gottenberg J.E.
      • et al.
      Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases.
      ]. The localization of B cells and their subsets were discovered in sarcoidosis lesions, in which naïve B cells and transitional B cells were increased concurrently with higher numbers of IL-10-producing regulatory B cells, compared with healthy controls, leading to abnormal B cell differentiation [
      • Saussine A.
      • et al.
      Active chronic sarcoidosis is characterized by increased transitional blood B cells, increased IL-10-producing regulatory B cells and high BAFF levels.
      ,
      • Celada L.J.
      • Drake W.P.
      Targeting CD4(+) T cells for the treatment of sarcoidosis: a promising strategy?.
      ]. Furthermore, our previous study showed increased B-cell-activating factor (BAFF) in the serum and skin lesions of sarcoidosis patients, in addition to abnormal B cell subsets [
      • Ueda-Hayakawa I.
      • et al.
      Elevated serum BAFF levels in patients with sarcoidosis: association with disease activity.
      ]. Therefore, dysregulated T and/or B cell populations and their aberrant function may induce sarcoidosis symptoms.
      Follicular helper CD4+ T (TFH) cells in humans were initially described in 2000 as a large proportion of CD4+ T cells in tonsils. They have a unique phenotype characterized by high levels of CXCR5 [
      • Crotty S.
      Follicular helper CD4 T cells (TFH).
      ,
      • Mesquita Jr., D.
      • et al.
      Follicular helper T cell in immunity and autoimmunity.
      ,
      • Deenick E.K.
      • Ma C.S.
      The regulation and role of T follicular helper cells in immunity.
      ]. The TFH programming is especially rich in cell surface receptor changes, making TFH cells relatively easy to be phenotypically distinguished into three subsets including Th1 (TFH 1), Th2 (TFH 2) and Th17 (TFH 17) based on their expression of CXCR3 and CCR6 receptors [
      • Nurieva R.I.
      • et al.
      Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages.
      ,
      • Yusuf I.
      • et al.
      Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).
      ,
      • Chtanova T.
      • et al.
      T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells.
      ,
      • Rasheed A.U.
      • et al.
      Follicular B helper T cell activity is confined to CXCR5(hi)ICOS(hi) CD4 T cells and is independent of CD57 expression.
      ]. This extensive set of cell surface receptor changes is reflective of the importance of cell-cell interactions between TFH and B cells [
      • Crotty S.
      Follicular helper CD4 T cells (TFH).
      ]. Furthermore, TFH cells are also important for the formation of germinal centers (GCs) [
      • Yusuf I.
      • et al.
      Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).
      ]. GCs are histologically distinct structures that develop within B cell zones of secondary lymphoid tissues, where the interrelated and multifaceted processes of B cell affinity maturation, class switch recombination, plasma cell differentiation, and memory B cell differentiation predominantly occur. That explains why TFH cells might play crucial roles in common autoimmune diseases; hence, they were reported as a potential biomarker in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) due to increased TFH cell numbers in those diseases [
      • Simpson N.
      • et al.
      Expansion of circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus.
      ,
      • King C.
      • Tangye S.G.
      • Mackay C.R.
      T follicular helper (TFH) cells in normal and dysregulated immune responses.
      ]. On the other hand, decreased TFH cells were found in ankylosing spondylitis, hepatocellular carcinoma and chronic infections [
      • Bautista-Caro M.B.
      • et al.
      Decreased frequencies of circulating follicular helper T cell counterparts and plasmablasts in ankylosing spondylitis patients Naive for TNF blockers.
      ,
      • Chowdhury A.
      • et al.
      Decreased T follicular regulatory cell/T follicular helper cell (TFH) in simian immunodeficiency virus-infected Rhesus macaques may contribute to accumulation of TFH in chronic infection.
      ,
      • Jia Y.
      • et al.
      Impaired function of CD4+ T follicular helper (Tfh) cells associated with hepatocellular carcinoma progression.
      ]. The features of TFH cells and their subsets in sarcoidosis patients have not yet been characterized. Therefore, we sought to investigate the expression of TFH cells, their subsets (TFH 1, TFH 2, TFH 17), and evaluate their correlation with Th17-associated cytokines in sarcoidosis patients.

      2. Materials and methods

      2.1 Ethics statement

      The study was performed under protocols reviewed and approved by the Ethic Committees on human experiments in Kansai Medical University, and all subjects gave informed consent prior to enrolling 2016155.

      2.2 Subjects

      All patients were admitted to the Department of Dermatology, Kansai Medical University Hospital from January to August 2017. The diagnosis of sarcoidosis in each patient was based on the guidelines of the American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Other Granulomatous Disorders statement [
      • van den Hoogen F.
      • et al.
      Classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative.
      ]. Complete medical histories, clinical examinations and laboratory tests were conducted for all patients at the first visit. Skin lesions, pulmonary involvement and eye involvement were assessed by biopsy; chest radiogram and high-resolution computed tomography; split-lamp and funduscopic examination, respectively. The panda sign and lambda sign as well as other involved organs were examined by imaging with 18F-fluorodeoxyglucose positron emission tomography and/or 67Ga scintigraphy uptake. Angiotensin-converting enzyme (ACE), soluble interleukin-2 receptor (sIL-2R), thymus and activation-regulated chemokine (TARC), and lysozyme were measured in the clinical laboratory. Peripheral blood was also obtained from 13 healthy subjects (3 male and 10 female) ages 58.25 ± 4.7 (mean ± SEM).

      2.3 Cell surface immunolabelling and flow cytometry

      The frequency and phenotype of circulating TFH and B cells were assessed by flow cytometry after staining 100 μL whole blood. Following incubation with Fc block (BD Bioscience, San Jose, CA, USA) at room temperature (RT) for 10 min, blood samples were stained with these surface antibodies for 15 m in. CD3-PerCP-Cy5.5, CD4-Pacific Blue, CD45RA-PE-Cy7, CXCR5-FITC, CCR6-PE, CXCR3-APC, CD19-FITC, IgD-PE, CD20-PE-Cy7, CD27-APC, CD38-Pacific Blue, and CD24-PerCP-Cy5.5. All antibodies were purchased from BD Bioscience. Red blood cells (RBC) from samples were then lysed by lysing solution (Beckman Coulter) and fixed in paraformaldehyde (PFA) for 10 min before washing in phosphate buffered saline (PBS). Cells were resuspended in 500 μL sheath buffer and analyzed with a FACS Canto II (BD Bioscience) using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

      2.4 Immunohistochemical staining

      Paraffin-embedded sections from skin biopsies were cut in 5 μm slices, deparaffinized, and rehydrated. A heat-induced epitope retrieval method was performed in Retrieval Solution pH 6 (Dako, Denmark) at 115 °C for 10 min. Tissues were treated with blocking one buffer (Nacalai Tesque, Inc., Japan) at RT for 1 h, incubated in the first antibody CXCR5 Abcam #46218, CD4 Leica Biosystem, UK or CD20 Cell Marque #120M-86 at RT for 2 h, CXCR3 Abcam #64714 and CCR6 Abcam #227036 at 37 °C for 1 h. After blocking endogenous peroxidase activity with 0.3 % H2O2, immunohistochemical detection was performed using the Envision + Duolink System-HRP (Dako) for CXCR5, CD4, CD20 primary antibodies and 2nd Horse anti-Mouse (Vector) or Rabbit (Vector) for CXCR3 and CCR6, respectively, at RT for 30 min. The reaction was developed with 3.3′ diaminobenzidine (DAB peroxidase substrate kit) for a maximum of 3 min. Sections were counterstained with hematoxylin and mounted. Section were double-stained with the first CD4 antibody (Leica Biosystem), then 2nd anti-Mouse IgG polyclonal antibodies (Nichirei Biosciences Inc. #414241), developed with ALP Muto Pure Chemicals Co. Ltd. #15682, and counterstained with the same CXCR5 antibody, then 2nd anti-Rabbit IgG polyclonal antibodies (Nichirei Biosciences Inc. #424144. All images were scanned using a NanoZoomer Hamamatsu Photonics, Japan.

      2.5 Isolation of CD4+CXCR5+ TFH cells from human peripheral blood

      Peripheral blood mononuclear cells (PBMCs) were separated immediately after blood sample collection using Ficoll-Paque Plus (GE Healthcare, Sweden). CD4+ T cells were purified from freshly isolated PBMCs by immunomagnetic negative selection using human CD4+ T cell isolation kits (Miltenyi Biotec #130-096-533) and a MACS separator, MACS LS columns (Miltenyi Biotec #130-042-401) and pre-separation filters (30 μm, Miltenyi Biotec). The CXCR5+ subpopulation was subsequently positively isolated from total CD4+ T cells using human CD185-APC (CXCR5) Ab (Miltenyi Biotec #130-098-422) and anti-APC microbeads (Miltenyi Biotec #130-090-855). Isolated T cells and TFH cells, >97 % and >90 % pure, respectively, were used immediately after isolation.

      2.6 Gene expression analysis by quantitative real-time PCR

      RNA was extracted and purified from total isolated TFH cells using RNA Plus XS kits NucleoSpin #740990, then reversed transcribed with SuperScript™ IV VILO™ Master Mix with ezDNase Invitrogen #11766050 to cDNA. A total of 25 μL of reaction sample was comprised of 20 ng of cDNA samples, primers, Rotor-Gene SYBR Green PCR Master Mix, and RNase free water (Rotor-Gene SYBR Green PCR Kit; Qiagen #204074. The specificity and PCR efficiency of each primer were confirmed by a single-peak melting curve analysis and standard curve of 5-fold serial dilutions; each individual sample was tested in triplicate and the results were quantified using the comparative 2−ΔΔCt method [
      • Schmittgen T.D.
      • Livak K.J.
      Analyzing real-time PCR data by the comparative CT method.
      ].

      2.7 Cytokine array assay

      The concentration of cytokines was measured by cytokine array assay. Serum was collected at the time of patients’ enrollment and stored at −20 °C until use. Bio-Plex Pro Human Th17 Cytokine Panel 15-plex (Bio-Rad #171AA001 M) and Bio-Plex Pro™ Human Inflammation Assays (Bio-Rad #171AL001 M) were performed following the manufacturer’s instructions and read on a Bio-Plex 200 System (Bio-Rad) with Bio-Plex manager software. Representative assay working range and assay sensitivity were based on standard curves prepared in Bio-Plex standard diluent HB.

      2.8 Statistical analysis

      All values with a normal distribution were shown as the mean and standard error of the mean (SEM) or standard deviation (SD). Student’s t-test and Mann Whitney U-tests were used to compare the frequencies of populations, mean or medians of different groups. Correlations were analyzed using Spearman’s and Pearson’s rank correlation coefficients. The results of quantitative real-time PCR were quantified using the comparative 2−ΔΔCt method. Nominal values were analyzed by chi-squared test. All P values less than 0.05 were defined as significant. All analyses were performed using SPSS statistics version 20 (IBM, New York, NY), FlowJo V10 (FlowJo, LLC, Ashland, OR, USA) and GraphPad Prism version 7 software (GraphPad Software, Inc., CA, USA).

      3. Results

      3.1 Clinical characteristics of patients with sarcoidosis

      This study enrolled twenty sarcoidosis patients (5 male and 15 female) from 42 to 81 years old. The clinical characteristics of patients with sarcoidosis are summarized in Table 1. All patients had skin lesions at the time of enrollment; but cutaneous symptoms were the only clinical manifestation in 25 % of patients. The clinical manifestations were different among the patients, in which 25 % presented with skin erythema, 25 % with skin papules, 20 % with skin nodules, 45 % with scar infiltrations, 10 % with cutaneous lichenoid and 15 % with subcutaneous lesions. Besides skin lesions, patients also showed other organ comorbidities, such as lung involvement (55 %) (Lambda sign was found on 82 % of them), eye involvement (55 %) and 25 % with one each of heart, joint, spleen or liver involvement.
      Table 1Clinical characteristics of the enrolled sarcoidosis patients.
      CharacteristicsSarcoidosis (n = 20)
      Age (years)61.95 (2.75)
      Male/Female5/15
      Duration of disease (years)4.05 (1.069)
      Number of involved organs2.7 (0.272)
      Affected organs
       Skin, n (%)20 (100 %)
       Lung, n (%)11 (55 %)
       Eyes, n (%)11 (55 %)
       Heart, n (%)5 (25 %)
       Joint, n (%)5 (25 %)
       Spleen, n (%)5 (25 %)
       Liver, n (%)5 (25 %)
      Laboratory marker
       ACE, U/L18.39 (1.82)
       Lysozyme, μg/mL10.635 (1.547)
       SIL-2R, U/mL725.8 (117.534)
       TARC, pg/μL1562.65 (278.28)
       CRP, mg/dL0.434 (0.216)
       WBC, %43.95 (4.1)
       Lymphocytes count, cells/μL1181.9 (127.39)
       Eosinophils, %2.925 (0.532)
       Monocytes, %6.895 (0.596)
      Data are mean and the standard error of the mean (SEM). TARC, thymus and activation-regulated chemokine; sIL-2R, soluble interleukin-2 receptor; ACE, angiotensin-converting enzyme; CRP, C-reactive protein; WBC, white blood cells.
.

      3.2 Detection of CD4+CXCR5+ TFH cell subsets in sarcoidosis patients

      We used flow cytometry to investigate circulating TFH cells and their subsets using samples from 20 patients and 13 healthy controls. We determined the frequency of the CD4+CXCR5+CD45RA TFH cell population, and identified three TFH subsets including CXCR3+CCR6 TFH 1, CXCR3CCR6 TFH 2 and CXCR3CCR6+ TFH 17 (Fig. 1A). The frequency of circulating TFH cells was significantly decreased (p < 0.001, Fig. 1B) while the mean fluorescence intensity (MFI) of CXCR5 was increased in sarcoidosis patients (p = 0.017, Fig. 1B). Among the three TFH subsets, the frequency of circulating TFH 1 did not differ between patients and controls (p = 0.393, Fig. 1C). However, there was a significant decrease in the frequency of circulating TFH 2 cells (p = 0.03, Fig. 1C) and an increase in the frequency of circulating TFH 17 cells in sarcoidosis patients (p = 0.008, Fig. 1C). These imbalanced populations may contribute to sarcoidosis pathogenesis. There was no correlation between the frequency of TFH cells or their subsets and disease activity in sarcoidosis patients (data not shown).
      Fig. 1
      Fig. 1Frequency of circulating TFH subsets in sarcoidosis patients and controls. Human peripheral blood was stained with superficial antibodies and analyzed by flow cytometry. A: TFH cells were identified by gating CD4+CD45CXCR5+ and 3 subsets were identified by CXCR3 and CCR6 antibodies. B: Circulating TFH cell frequency was significantly decreased, while the mean fluorescence intensity of CXCR5 expression was increased in sarcoidosis patients. C: The frequency of circulating TFH 1 did not differ, while there was a significant decrease of circulating TFH 2 frequency and an increase in circulating TFH 17 frequency in sarcoidosis patients. *p < 0.05, **p < 0.01, ***p < 0.001, ns: difference not significant.

      3.3 TFH cells migrated into affected tissues in sarcoidosis

      Skin samples from cutaneous sarcoid lesions were collected randomly from 5 patients and CXCR5- and CD4-positive cells were found in all biopsy tissues in serial slide sections (Fig. 2A, B). Furthermore, we checked double-staining of CD4 and CXCR5 in our patients’ tissues and found the double-positive cells, as shown in Supplementary Fig. 1. The histopathological analysis showed that “mature stage” granulomas with sparse inflammatory infiltration had small numbers of TFH cells (Fig. 2A.2 and B.2), whereas “early stage” granulomas contained many inflammatory cells admixed with granulomas expressing large numbers of TFH (Fig. 2A.1 and B.1). To exclude the possibility that CXCR5+ cells could be B cells, we performed immunohistochemical staining for CD20. As shown in Fig. 2C, there were B cells that had migrated into sarcoidosis lesions, but the position of them did not coincide with CXCR5-positive cells (Fig. 2C, C.1, C.2). In addition, the immunohistochemical staining with CXCR3 and CCR6 antibodies showed many CXCR3 and CCR6-positive cells in sarcoidosis skin lesions (Supplementary Fig. 2A).
      Fig. 2
      Fig. 2TFH cells migrated into affected tissues in sarcoidosis. IHC staining of a representative sarcoidosis lesion showed positive CD4 (A), CXCR5 (B), CD20 (C) cells around the granulomas (scale bar = 100 μm). “Early stage” granulomas contained many inflammatory cells admixed with granulomas containing large numbers of CXCR5-positive cells (B.1), whereas “mature stage” granulomas with sparse inflammatory infiltration had small numbers of CXCR5-positive cells (B.2). Intense CD4 T cells presented in the same tissue locations (A.1 and A.2), while CD20 – B cells appeared in different locations with CXCR5-positive cells in sarcoidosis lesions (C.1 and C.2).
      To distinguish TFH subsets, we stained patients’ skin tissues with differentially fluorescence-labeled antibodies specific for CXCR5, CXCR3, and CCR6. As shown in Supplementary Fig. 2B, we found that most of CXCR5-positive cells exhibited positive for CXCR3, indicating that CXCR5+ cells are at least in part composed of TFH 1 subset. In spite of the abundant CCR6+ cells by immunohistochemistry (Supplementary Fig. 2A), we failed to detect CCR6 by immunofluorescent staining, although we have been tried several antibodies and staining protocols for CCR6.

      3.4 TFH cells expressed higher levels of Th17 cytokines and lower levels of Th1 and Th2 cytokines

      In order to check the function of TFH cells, we isolated them from PBMC and analyzed gene expression by quantitative real-time PCR. T cells and TFH cell subpopulations, >97 % and >90 % pure, respectively, were used immediately after isolation (Fig. 3A). Although sarcoidosis is known as a Th1 disease characterized by the presence of IFN-γ in affected tissues and during granuloma formation, expression of IFN-γ was decreased in TFH cells from sarcoidosis patients, in addition to lower levels of IL-4 (p = 0.005 and p = 0.029, respectively, Fig. 3B).
      Fig. 3
      Fig. 3TFH cells expressed higher levels of Th17 cytokines, and lower levels of Th1 and Th2 cytokines in sarcoidosis. A: Isolation of CD4+CXCR5+ TFH cells from human peripheral blood. Isolated T cells and TFH cells were >97 % and >90 % pure, respectively, and used immediately after isolation. B: Gene expression by quantitative real-time PCR in isolated TFH cells (each patient and control group, n = 3, tested in triplicate). Expression of IFN-γ, representative Th1 cytokines and IL-4 indicative of Th2 were significantly lower in sarcoidosis TFH cells compared with those in the control group (p = 0.005 and p = 0.029). mRNA expression of IL-17A, representative for Th17, and programmed cell death-1 (PD-1) were both upregulated in our patients (p = 0.003 and p = 0.01, respectively). C: Th17 cytokines in patients’ sera including IL-17A, IL-17 F and TNF-α, were increased, as evaluated by cytokine array assay (p = 0.005, p = 0.001, p < 0.001, respectively). *p < 0.05, **p < 0.01, ***p < 0.001, ns: difference not significant.
      Interestingly, levels of IL-17A and programmed cell death-1 (PD-1) were upregulated in our patients (p = 0.003 and p = 0.01, respectively, Fig. 3B). Moreover, a cytokine array assay revealed an increase in Th17 cytokines in patients’ sera, including IL-17A, IL-17 F and TNF-α (p = 0.005, p = 0.001, p < 0.001, respectively, Fig. 3C). However, we could not find any correlations between IL-17 gene expression in TFH cells and serum Th17 cytokines levels.

      3.5 No correlation between gene expression of IL-21, CD40 L, IL-6 in TFH cells and serum concentration of these cytokines in sarcoidosis

      It is known that TFH cells selectively express a wealth of surface proteins to assist with localization and direct physical interactions with B cells in order to provide B cell help. IL-21, CD40 L and IL-6 are important cytokines for B cell activation, proliferation and differentiation. These cytokines were significantly increased in sarcoidosis patients’ sera (p = 0.001, p < 0.001 and p = 0.007, respectively, Fig. 4A). However, gene expression of IL-21, CD40 L and IL-6 in TFH cells did not differ compared with healthy controls (Fig. 4B).
      Fig. 4
      Fig. 4No correlation between gene expression of IL-21, CD40 L, IL-6 in TFH cells and serum concentrations of these cytokines in sarcoidosis. A: These cytokines were significantly increased in sarcoidosis patients’ sera determined by cytokine array assay (p = 0.001, p < 0.001 and p = 0.007). B: Gene expression by quantitative real-time PCR in isolated TFH cells (each patient and control group, n = 3, tested in triplicate) showed no difference between the groups. **p < 0.01, ***p < 0.001, ns: difference not significant.

      3.6 Peripheral B cell profile in sarcoidosis patients

      TFH cells are required for the formation and maintenance of GCs and the generation of most memory B cells and plasma cells. This is a complex process and the mechanisms involved are not completely understood. In this study, we confirmed the upregulation of B cell activation factor from TNF family (BAFF) and IL-10 (p < 0.001 and p = 0.016, Fig. 5A). To identify B cell subsets, we collected lymphocytes, CD24+CD38+ transitional B cells and CD19+ B cells. Then, from the B cells subset, we detected CD20CD38+ plasmablasts, CD20+CD27IgD+ naïve B cells, and CD20+CD27+ memory B cells in which CD20+CD27+IgD+ pre-switch memory B cells and CD20+CD27+IgD post-switch memory B cells were identified (Supplementary Table 1 and Fig. 5B). The frequency and MFI of CD38 in the plasma cell subset did not differ in comparison with control group (p = 0.73 and p = 0.75, Fig. 5C). However, there was a significant increase in transitional B cells and naïve B cell frequencies (p = 0.001 and p < 0.001, respectively, Fig. 5C), and a significant decrease in the memory B cells frequency (p < 0.001, Fig. 5C) that paralleled with the pre-switch memory B cells, while post-switch memory B cells were not different in sarcoidosis patients. The MFI of CD20, CD27, CD38 and IgD in these subsets was also analyzed without any significant differences between the groups (Supplementary Table 1). Although we observed the altered B cell subsets in sarcoidosis patients, we could not find any association between B cell alteration and the imbalance of TFH cells.
      Fig. 5
      Fig. 5B cell differentiation without the regulation of TFH cells in sarcoidosis disease. A: upregulation of B cell activation factor from TNF family (BAFF) and IL-10 in sarcoidosis patients (p < 0.001 and p = 0.016). B: Detection of B cell subsets by superficial antibodies staining and flow cytometry. We detected CD24+CD38+ transitional B cells, CD20CD38+ plasma cells, CD20+CD27IgD+ naïve B cells, and CD20+CD27+ memory B cells in which CD20+CD27+IgD+ pre-switch memory B cells and CD20+CD27+IgD post-switch memory B cells were found. C: We found a significant increase of transitional and naïve B cell frequencies (p = 0.001 and p < 0.001, respectively), and a decrease in memory B cell frequency (p < 0.001), while plasma cell subsets did not show any differences compared with control. *p < 0.05, ***p < 0.001, ns: difference not significant.

      4. Discussion

      In our study, the frequency of circulating TFH cells was significantly decreased while the MFI of CXCR5 expression was increased in sarcoidosis patients (Fig. 1B). It is known that one of the main immunologic features of sarcoidosis is the influx of CD4+ T cells into the active sites of this disease. TFH cells are non-polarized CD4+ T cells that express high levels of the chemokine receptor CXCR5; the homing exerted by its ligand CXCL13 chemokine allows follicular migration, where they are thought to act as a regulator of host humoral immune responses [
      • Chowdhury A.
      • et al.
      Decreased T follicular regulatory cell/T follicular helper cell (TFH) in simian immunodeficiency virus-infected Rhesus macaques may contribute to accumulation of TFH in chronic infection.
      ,
      • Cagigi A.
      • et al.
      Altered expression of the receptor-ligand pair CXCR5/CXCL13 in B cells during chronic HIV-1 infection.
      ]. Infiltration of TFH into skin lesions (Fig. 2 and Supplement Fig. 2) suggests circulating TFH cells, at least in part TFH 1, are recruited to affected tissues, and localized to the periphery of new and early granulomas, where they fulfill their inflammatory role in sarcoidosis.
      Recently, the pro-inflammatory cytokine IL-17A has been implicated in the pathogenesis of many autoimmune diseases such as RA, SLE, multiple sclerosis, and sarcoidosis. IL-17A was expressed in sarcoid tissues and present in lung tissues, localizing around and inside granulomas in response to chemotactic chemokines [
      • Ten Berge B.
      • et al.
      Increased IL-17A expression in granulomas and in circulating memory T cells in sarcoidosis.
      ,
      • Facco M.
      • et al.
      Sarcoidosis is a Th1/Th17 multisystem disorder.
      ]. Therefore, the immunopathology of sarcoidosis is now recognized as a combination of Th1 and Th17 reactions. The TFH 17 subset was also evaluated and demonstrated to correlate with disease activity and therapeutic response in other autoimmune diseases, including RA and juvenile dermatomyositis [
      • Singh D.
      • et al.
      Analysis of CXCR5(+)Th17 cells in relation to disease activity and TNF inhibitor therapy in rheumatoid arthritis.
      ,
      • Morita R.
      • et al.
      Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.
      ]. In our study, we found that the TFH 17 subset was upregulated, while TFH 1 resembled that in healthy controls. In addition, isolated circulating TFH cells from patients showed an upregulation of IL-17A as well as PD-1, indicative of TFH 17 (Fig. 3B). However, we did not identify any correlation between the TFH 17 subset and sarcoidosis disease activity or Th17 cytokines, as well as correlations between IL-17 mRNA expression in TFH cells and serum Th-17 cytokine levels. These results may possibly be explained by the small numbers of patients, or that IL-17 is produced by many potential cells via various pathways and interactions besides TFH cells in this disease.
      The avoidance of autoimmunity is heavily dependent on T and B cell tolerance mechanisms. The shift toward a TFH 17-dominant immune response is essential for regulating B cells activation. Recently, the potential roles for IL-17A and TFH 17 cells subset in GC are gaining special attention. Yanna Ding et al. indicated that even in normal mice, IL-17 signaling can regulate TFH cell localization in GC light zone via upregulation of regulator of G protein signaling in TFH cells to promote GC development and high affinity antibody production [
      • Ding Y.
      • et al.
      IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.
      ]. Furthermore, the TFH 17 cell subset was demonstrated to help B cell differentiation by inducing naïve B cells, plasmablasts and promoting class switching [
      • Singh D.
      • et al.
      Analysis of CXCR5(+)Th17 cells in relation to disease activity and TNF inhibitor therapy in rheumatoid arthritis.
      ,
      • Morita R.
      • et al.
      Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.
      ]. We evaluated B cell differentiation by analyzing the B cell subset profile. Our data showed that there is no difference in frequency of the plasmablasts, while the proportion of memory B cells, naïve and transitional B cells changed, as shown in Fig. 5. As previously reported, abnormal B cell differentiation was found in sarcoidosis and corresponded to increased levels of important cytokines including IL-21, CD40 L and IL-6 in this disease (Fig. 4A) [
      • Shapiro-Shelef M.
      • Calame K.
      Regulation of plasma-cell development.
      ,
      • Chattopadhyay P.K.
      • Yu J.
      • Roederer M.
      A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles.
      ,
      • Arpin C.
      • et al.
      Generation of memory B cells and plasma cells in vitro.
      ]. IL-21 is a potent inducer of plasma cell differentiation, particularly when CD40 L signals are reduced. Numerous other cytokines enhance plasma cell differentiation, including IL-10, IL-6, and IL-4 [
      • Shapiro-Shelef M.
      • Calame K.
      Regulation of plasma-cell development.
      ]. Although TFH cells are critical for the development and maintenance of GCs via molecules such as CD40 L and IL-21 which are thought to be a key mechanism of selection of high-affinity B cells in various autoimmune diseases, there was no correlation between gene expression of these cytokine levels in isolated TFH cells and their concentration in patients’ sera. Therefore, it is difficult to determine the contribution of TFH cells to B cell differentiation. One possibility is that other cells, not circulating TFH, produced those proteins and induced B cell differentiation or that a complex immune network may be necessary in inducing the aberrant cytokine production. Another possibility is that in this study we only examined the circulating TFH, but not total TFH cells in patients, so the results might not be representative for TFH cells in sarcoidosis.
      Circulating TFH cells have been found to be associated with the development of many autoimmune diseases and chronic inflammatory skin diseases [
      • Gensous N.
      • et al.
      T follicular helper cells in autoimmune disorders.
      ]. Chang et al. reported that aggregates of T and B cells were observed in lupus nephritis tissue, and they clustered with B cells building structures similar to ectopic germinal centers (GCs), suggesting organ-intrinsic adaptive immune responses in the pathogenesis of lupus nephritis [
      • Chang A.
      • et al.
      In situ B cell-mediated immune responses and tubulointerstitial inflammation in human lupus nephritis.
      ]. As we found substantial numbers of CXCR5+ CD4+ TFH cells in affected tissues, they might have important roles in providing help to co-resident B cells in situ, although the affected tissue from sarcoidosis patients does not exhibit GC formation. Moreover, it is also possible that they fulfill their inflammatory role by producing cytokines in the tissue. To distinguish TFH subsets within the skin lesion, we stained patients’ skin tissues with differentially-labeled antibodies specific for CXCR5, CXCR3, and CCR6. As shown in Supplementary Fig. 2B, we found that most of CXCR5-positive cells exhibited positive for CXCR3, indicating that CXCR5+ cells are at least in part composed of TFH 1 subset. Further study is required to elucidate accurate TFH subsets within skin tissue of sarcoidosis.
      In conclusion, we have herein described a decrease of circulating TFH cells that may migrate to inflamed tissues, localizing around granulomas. We also identified the upregulation of the TFH 17 cells subset together with an increase in Th17 cytokine levels that may help induce an inflammatory cascade in sarcoidosis. Although we could not demonstrate a direct linkage between the imbalance of TFH cells and abnormal B cell differentiation, this first overall view for TFH cell expression in sarcoidosis will hopefully spur further insights in the complicated pathogenic mechanisms of this disease.

      Declaration of Competing Interest

      The authors have no conflict of interest to declare.

      Acknowledgment

      We thank all the patients who agreed to take part in our study.

      Appendix A. Supplementary data

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