Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease This information is current as of September 15, 2017.
Ryuta Kamekura, Kenichi Takano, Motohisa Yamamoto, Koji Kawata, Katsunori Shigehara, Sumito Jitsukawa, Tomonori Nagaya, Fumie Ito, Akinori Sato, Noriko Ogasawara, Chieko Tsubomatsu, Hiroki Takahashi, Hiroshi Nakase, Tetsuo Himi and Shingo Ichimiya
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J Immunol published online 15 September 2017 http://www.jimmunol.org/content/early/2017/09/15/jimmun ol.1601507
Published September 15, 2017, doi:10.4049/jimmunol.1601507
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Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease Ryuta Kamekura,*,†,1 Kenichi Takano,†,1 Motohisa Yamamoto,‡ Koji Kawata,* Katsunori Shigehara,* Sumito Jitsukawa,*,† Tomonori Nagaya,† Fumie Ito,*,† Akinori Sato,* Noriko Ogasawara,† Chieko Tsubomatsu,† Hiroki Takahashi,‡ Hiroshi Nakase,x Tetsuo Himi,† and Shingo Ichimiya*
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mmunoglobulin G4–related disease (IgG4-RD) shows a chronic fibroinflammatory condition characterized by spread to various systemic organs, resulting in dysfunctions associated with IgG4 such as dacryoadenitis and sialadenitis (also so-called Mikulicz’s disease) and type 1 autoimmune pancreatitis. As named, IgG4-RD commonly shows elevated serum IgG4 concentrations and typical pathological findings including marked infiltration of IgG4+ plasma cells, storiform fibrosis, and obliterative phlebitis in *Department of Human Immunology, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan; † Department of Otolaryngology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan; ‡Department of Rheumatology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan; and xDepartment of Gastroenterology and Hepatology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan 1
R.K. and K.T. contributed equally to this study.
ORCIDs: 0000-0001-8706-6384 (K.T.); 0000-0003-2006-1751 (M.Y.); 0000-00026688-1993 (F.I.); 0000-0003-2848-6586 (H.N.). Received for publication September 13, 2016. Accepted for publication August 28, 2017.
the involved organs (1). Treatment with glucocorticoids is effective for IgG4-RD; however, relapse frequently occurs after tapering or discontinuing administration of glucocorticoids (2). Recent studies have shown that rituximab, an antiCD20 Ab that depletes B cells, is also effective in some cases of IgG4-RD (3), suggesting that defects of humoral immunity mediated by T and B lymphocytes underlie the pathogenesis of IgG4-RD. Serum IgG4 level has been used as a diagnostic marker of IgG4-RD. However, clinical significance of serum IgG4 level as a biomarker for IgG4-RD has been debated because of high false-positive and -negative rates (4, 5) and a high relapse rate (2). Recently, Wallace et al. (6) reported that plasmablasts in peripheral blood are markedly increased in patients with active IgG4-RD, and that plasmablast count is a useful biomarker for IgG4-RD. Efforts are currently being made to find an additional clinical marker for IgG4-RD for the development of a treatment strategy. The pathogenesis of IgG4-RD also remains controversial. There are several reports showing findings consistent with an autoimmune disorder (7), an allergic disorder (8), and an infection (9) underlying IgG4-RD. Ectopic germinal centers (GCs) are frequently observed in lesions of IgG4-RD (1), indicating aberrant activation of humoral immune responses in IgG4-RD. To establish Ag-specific humoral immunity, B cells form GCs of lymphoid follicles in concert with T follicular helper (Tfh) cells, which are a class of effector helper CD4+ T cells and share CXCR5 with B cells (10). Tfh cells primarily localize and work in secondary lymphoid organs, such as the spleen, lymph nodes, and tonsils, but they are also found in peripheral blood and lesions of diseases (11). In lymphoid organs, GC-Tfh cells, which express high levels of Tfh cell–related molecules such as B cell lymphoma (BCL) 6, This work was supported by Japanese Society for the Promotion of Science Grants 15K10787 (to R.K.), 15K10818 (to K.T.), 15K20214 (to K.K.), 15K16399 (to A.S.), 15K20213 (to C.T.), and 26293370 and 16K15723 (to T.H.). Address correspondence and reprint requests to Dr. Ryuta Kamekura, Department of Human Immunology, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BCL, B cell lymphoma; cTfh, circulating Tfh; GC, germinal center; IgG4-DS, IgG4-related dacryoadenitis and sialadenitis; IgG4-RD, IgG4-related disease; PD-1, programmed death 1; SMG, submandibular gland; SS, Sjo¨gren’s syndrome; Tfh, T follicular helper. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00
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IgG4-related disease (IgG4-RD) is a newly recognized systemic chronic fibroinflammatory disease. However, the pathogenesis of IgG4-RD remains unknown. To determine the pathophysiologic features of IgG4-RD, we examined T follicular helper (Tfh) cells in lesions and blood from patients with IgG4-RD. Patients with IgG4-related dacryoadenitis and sialadenitis (IgG4DS) showed increased infiltration of Tfh cells highly expressing programmed death 1 and ICOS in submandibular glands. Tfh cells from IgG4-DS submandibular glands had higher expression of B cell lymphoma 6 and a greater capacity to help B cells produce IgG4 than did tonsillar Tfh cells. We also found that the percentage of programmed death 1hi circulating Tfh cells in IgG4-DS patients was higher than that in healthy volunteers and was well correlated with clinical parameters. Our findings indicate that anomalous Tfh cells in tissue lesions of IgG4-RD have features distinct from those in lymphoid counterparts or blood and potentially regulate local IgG4 production in IgG4-RD. The Journal of Immunology, 2017, 199: 000–000.
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CUTTING EDGE: LESIONAL Tfh CELLS IN IgG4-RELATED DISEASE Abs For flow cytometry, the following anti-human mAbs and isotype-matched control IgG were purchased from BD Biosciences (San Jose, CA): mouse anti–CD3-allophycocyanin (UCHT1), anti–CD4-allophycocyanin-Cy7 (RPAT4), anti–CD8-PE (Leu-2a), anti–CD19-allophycocyanin-Cy7 (SJ25C1), anti– CD27-FITC (M-T271), anti–PD-1-PE (EH12.1), anti–ICOS-BV421 (DX29), and anti–BCL6-PE (K112-91) mAbs and a rat anti–CXCR5-PerCP-Cy5.5 (RF8B2) mAb. A mouse anti-human CD38-BV421 (HIT2) mAb was obtained from BioLegend (San Diego, CA). For immunofluorescence microscopy, we used mouse anti-human BCL6 (LN22), CD4 (1F6), and IgG4 (HP6025) mAbs purchased from Nichirei (Tokyo, Japan).
FACS analysis and cell sorting PBMCs were isolated from heparinized blood specimens by centrifugation over a discontinuous density gradient (Lympholyte-H; Cedarlane, Burlington, ON, Canada). Tissue samples of SMGs and tonsils were mechanically disrupted into single-lymphocyte suspensions for flow cytometry and cell sorting. Cell staining using cell surface markers was performed as previously described (19). Intracellular staining for BCL6 was performed with Transcription Factor Buffer Set (BD Biosciences) as described in the protocol of the manufacturer. Samples were then analyzed and sorted using a FACSCanto II and FACSAria II, respectively (BD Biosciences). In each experiment using FACS-sorted cells, the purity of cells reached 95% after validation with reanalysis using FACSCanto II. All data were analyzed using FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).
Quantitative real-time PCR Quantitative real-time PCR was performed using a TaqMan Gene Expression Assay kit (Life Technologies, Carlsbad, CA) with the Roche LightCycler 480 Real-Time PCR Detection System (Roche Diagnostics, Mannheim, Germany) as described in the protocol of the manufacturer. For TaqMan-based detection, the amount of GAPDH mRNA was used to standardize the amounts of target transcripts including BCL6 (Hs00153368), CXCL13 (Hs00757930), IL-4 (Hs00174122), IL-10 (Hs00961622), and IL-21 (Hs00222327). The DD cycle threshold method was used to calculate the relative mRNA expression of triplicate specimens.
Immunofluorescence microscopy
Materials and Methods Study populations Characteristics of patients with IgG4-DS (n = 42), Sjo¨gren’s syndrome (SS; n = 16), and atopic asthma (asthma; n = 20) and healthy volunteers (n = 36) are summarized in Supplemental Table I. Diagnosis of IgG4-DS was performed according to the 2011 comprehensive IgG4-RD diagnostic criteria (16). Involved organs in patients with IgG4-DS are summarized in Supplemental Table II. At the first blood sampling, none of the patients had received glucocorticoid therapy. Treatment for IgG4-DS was performed according to our protocol as previously described (2). Patients with primary SS were diagnosed in accordance with the revised European criteria (17). The diagnosis of asthma was established on the basis of the Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma 2015 and Japan Respiratory Society Guidelines (18). Subjects with asthma were diagnosed by questionnaires, physical examination, spirometry, airway reversibility and hyperreactivity tests, measurement of specific IgE, and fractional exhaled NO. No patient had a history of allergen-specific immunotherapy. Before entrance to treatment protocols, blood specimens were collected from the subjects and analyzed. None of the healthy volunteers had abnormal physical or chest x-ray findings, and results of all blood tests were negative. All subjects were nonsmokers. Written, informed consent was obtained in all cases according to the Declaration of Helsinki. All protocols were approved by the Institutional Review Board of Sapporo Medical University Hospital.
Tissue specimens Tissues of the SMG, nasal polyps, and palatine tonsils were obtained from six patients with IgG4-DS (donor age range: 52–72 y), three patients with chronic rhinosinusitis (donor age range: 43–50 y), and six patients with tonsillar hypertrophy (donor age range: 31–54 y) for diagnosis or treatment at Sapporo Medical University Hospital. Normal SMG tissues were obtained from six patients with head and neck cancer (donor age range: 54–79 y) who had undergone supraomohyoid neck dissection.
For immunostaining of tissues of SMGs, 10-mm-thick frozen sections were made with a cryostat. The tissue sections were fixed with ice-cold acetone and ethanol (1:1) for 10 min. After rinsing in PBS, the tissue sections were incubated with primary Abs at room temperature for 1 h. Subsequently, the tissue sections were incubated with Alexa 488 (green)–conjugated antirabbit or Alexa 594 (red)–conjugated anti-mouse IgG (Life Technologies) at room temperature for 1 h. Then nuclei were stained using DAPI (Dojindo Laboratories, Kumamoto, Japan) in PBS at room temperature for 15 min. Some tissue sections of SMGs were stained with H&E (Sakura Finetek Japan, Tokyo, Japan). The specimens were examined and photographed with an LSM780/ELYRAS.1 microscope (Carl Zeiss, Oberkochen, Germany).
Measurement of Igs Concentrations of IgG1 and IgG4 in cell-free culture supernatants were analyzed in triplicate with ELISAs as described in the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI). The detection limits of IgG1 and IgG4 are 3.13 and 7.8 ng/ml, respectively.
B cell coculture assays Sorted Tfh (CD3+CD4+CXCR5+PD-1hi) cell subsets from SMGs, tonsils, or blood specimens were plated with blood allogeneic or autologous B cells (CD32CD19+) from healthy volunteers in a 1:1 ratio and cocultured for 12 d in the presence of 2.5 mg/ml staphylococcal enterotoxin B (SigmaAldrich, St. Louis, MO). Supernatants were analyzed by ELISA.
Statistical analysis All data are shown as mean 6 SD. Significant differences between any two groups were determined by using Student t test or the Mann–Whitney U test. Multiple-group comparisons were analyzed with one-way ANOVA or the Kruskal–Wallis test. Correlations were determined by Pearson or Spearman correlation coefficients. The p values ,0.05 were considered significant.
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programmed death 1 (PD-1), and ICOS, help neighboring GC-B cells to undergo class-switch recombination and affinity maturation (10). In contrast, Tfh cells in peripheral blood are recognized as memory Tfh cells (12, 13). Such circulating Tfh (cTfh) cells also have an intrinsic ability to provide B cell help in vitro despite having lower BCL6 expression than that in Tfh cells residing in lymphoid tissues (12, 14). We have recently reported that cTfh cells have pathological relevance to allergic airway diseases such as allergic rhinitis and bronchial asthma, and that they are potentially related to the development of allergic airway diseases (15). In this study, we first showed that a large number of Tfh (CD3+CD4+CXCR5+) cells characterized by high expression levels of BCL6, PD-1, and ICOS had infiltrated submandibular glands (SMGs) from patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) compared with SMGs from patients with head and neck cancer as controls. Functional analyses further revealed that Tfh cells in SMGs from IgG4-DS patients had a higher capacity than tonsillar Tfh cells to help B cells produce IgG4. We also found that IgG4-DS patients showed large numbers of PD-1hi cTfh cells and that their percentage was positively correlated with serum level of IgG4, ratio of IgG4 to total IgG (IgG4/IgG ratio), and number of involved organs in IgG4-DS patients. In addition, clinical remission achieved by treatment with glucocorticoids was associated with a reduction in the number of PD-1hi cTfh cells. Collectively, our findings indicate that Tfh cells in tissue lesions of IgG4-RD have features that are distinct from those in lymphoid counterparts or blood, and that the cells potentially regulate local IgG4 production in IgG4RD. This study provides novel insights into the role of activated Tfh cells in the pathogenesis of IgG4-RD.
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Results and Discussion hi hi
Functional PD-1 ICOS Tfh cells abundantly infiltrate tissue lesions of IgG4-DS
showed abundant infiltration of CD4+BCL6+ cells in submandibular sections from patients with IgG4-DS (Fig. 1D). In addition, we found that such Tfh cells concomitantly expressed PD-1 and ICOS at high levels compared with those in control SMGs, nasal polyps, tonsils, and peripheral blood (Fig. 1E). It was noted that ICOShi Tfh cells localizing in IgG4-DS SMGs distinctively expressed PD-1 at a marked level, and that such a population of Tfh cells highly expressing PD-1 was not found in other tissues including blood (Fig. 1F). Next, we sought to characterize Tfh cells in SMGs from patients with IgG4-DS. Quantitative real-time PCR analyses of functional molecules in general Tfh cells demonstrated that Tfh cells in IgG4-DS SMGs abundantly possessed transcripts encoding BCL6, CXCL13, and IL-10, whereas the amounts of transcripts encoding IL-4 and IL-21 were comparable with those in tonsils and blood (Fig. 2A). Investigation of BCL6 expression at the protein level by intracellular flow cytometry revealed that Tfh cells in IgG4-DS SMGs indeed had a larger amount of BCL6 than did Tfh cells in tonsils and blood (Fig. 2B). Collectively, the results suggested unequivocal
FIGURE 1. Abundant infiltration of PD-1hiICOShi Tfh cells in tissue lesions of IgG4-DS. (A) Results of immunofluorescence confocal microscopy showed the expression of IgG4, CD3, and CD4 in a normal human SMG (control SMG) and an SMG from a patient with IgG4-DS SMG. Nuclei were stained using DAPI. Red (middle panel), IgG4; green, CD3; red (lower panel), CD4; blue, DAPI. Consecutive sections of normal and IgG4-DS SMG tissues were stained with H&E (upper panel). (B) Representative FACS profiles indicating CD4+ T cells (CD3+CD4+) and CD8+ T cells (CD3+CD8+). Plots were pregated on CD3+ cells and examined by the levels of CD4 and CD8. Numbers indicate percentages of cells in the gate. Percentages of CD4+ and CD8+ T cells in CD3+ cells from IgG4-DS SMG (black bars) and control SMG (white bars) are shown. Results are expressed as mean 6 SD of four independent experiments. (C) Representative FACS profiles indicating CXCR5+ cells in CD4+ T cells (CD3+CD4+). Plots were pregated on CD3+CD4+ cells and examined by the level of CXCR5. Percentages of CD3+CD4+CXCR5+ cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. (D) Results of immunofluorescence confocal microscopy showed the expression of CD4 and BCL6 in a normal human SMG (control) and an SMG from a patient with IgG4-DS (IgG4-DS). Green, CD4; red, BCL6. (E) Representative histograms showing the expression of PD-1 and ICOS in CD4+CXCR5+ cells from IgG4-DS SMG, control SMG, nasal polyp, tonsil, and normal blood. Numbers indicate percentages of cells in the gate. Percentages of PD-1hiICOShi cells in CD4+CXCR5+ cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. (F) Mean fluorescence intensities (MFIs) of PD-1 in ICOShi Tfh cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. Scale bars, 20 mm. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
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Ectopic GCs are frequently observed in the lesions of IgG4-RD (1), indicating infiltration of lymphocytes and aberrant activation of humoral immune responses in IgG4-RD. Therefore, we first examined histopathological features of SMGs in IgG4-DS. As shown in Fig. 1A, dense infiltration of CD3+ CD4+ T cells, as well as IgG4+ cells, was observed in SMGs from patients with IgG4-DS compared with SMGs from controls. We also found a profile of CD4+ T cells dominantly infiltrating SMGs from patients with IgG4-DS by flow cytometry (Fig. 1B). A previous study suggested that Tfh cells, which are characterized by expression of CXCR5 and are a major source of IL-21, contribute to GC formation and IgG4 production in IgG4-RD (20). We therefore examined CD4+ CXCR5+ T (Tfh) cells in SMGs from patients with IgG4-DS. Interestingly, the majority of CD4+ T cells infiltrating IgG4DS SMGs were Tfh cells, accounting for 72.3% of CD4+ T cells (Fig. 1C). This finding was further supported by immunofluorescence labeling confocal microscopy that
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functions of Tfh cells in the pathogenesis of IgG4-DS. Further functional analyses revealed that Tfh cells in IgG4-DS SMGs had a higher capacity than that of tonsillar Tfh cells to help B cells produce IgG1 and IgG4 (Fig. 2C). Expression level of BCL6 reflects the developmental state of Tfh cells, and repeated encounters with APCs reinforce BCL6 expression in Tfh cells (21). Although the precise origin of Tfh cells residing in IgG4-DS SMGs remains unknown, our findings indicate that such Tfh cells in IgG4-DS might have many opportunities to interact with APCs and subsequently acquire specialized features to produce IgG4. The mechanisms of class switching to IgG4 remain unknown. To eliminate the effects of B cells from different donors, we performed coculture of Tfh cells from IgG4-DS SMGs or tonsils with allogenic blood B (CD32CD19+) cells from healthy volunteers. Unfortunately, we could not use naive or unswitched memory B cells because of the difficulty in accessing additional samples from patients with IgG4-DS. Therefore, this experiment will be performed in a future study to clarify the different helper activity from Tfh cells. Clinical relevance of PD-1hi cTfh cells in IgG4-DS
Blood circulation is one of the main reservoirs of functional Tfh cells (12). We therefore examined PD-1hi cTfh (CD3+ CD4+CXCR5+PD-1hi) cells in peripheral blood from patients with IgG4-DS. In general, cTfh cells include cells expressing PD-1, which represent an active form of circulating memory Tfh cells (13). We initially examined the percentage and absolute number of PD-1hi cTfh cells in total cTfh cells from patients with IgG4-DS by flow cytometry (Fig. 3A). We also analyzed cTfh cells in patients with SS and patients with asthma for comparison with those in patients with IgG4-DS. The patients with IgG4-DS showed a much larger proportion and number of PD-1hi cTfh cells in total cTfh cells than those in healthy volunteers (Fig. 3A). The patients with SS also showed a high proportion, but not number, of PD-1hi cTfh cells. In contrast, the proportion and number of PD-1hi cTfh
cells in patients with asthma were not significantly different from those in healthy volunteers. To further understand the clinical relevance of PD-1hi cTfh cells in IgG4-RD patients, we examined the percentage of PD-1hi cTfh cells in IgG4-DS patients with or without multiple involvement of other organs in addition to lacrimal and/or salivary glands (comorbidities). As shown in Fig. 3B, the percentage of PD-1hi cTfh cells was higher in IgG4-DS patients with comorbidities. We also studied the relationships between percentage of PD-1hi cTfh cells and chief clinical findings including serum IgG4 level, IgG4/IgG ratio, and number of involved organs as diagnostic parameters (Fig. 3C). Notably, the percentage of PD-1hi cTfh cells was significantly correlated with serum level of IgG4 (r = 0.5484, p = 0.0004), IgG4/IgG ratio (r = 0.4269, p = 0.0084), and number of involved organs (r = 0.6305, p , 0.0001). Similar results were obtained in a previous study by Akiyama et al. (22). To examine the functional properties of PD-1hi cTfh cells in IgG4DS patients, we cocultured PD-1hi cTfh cells with autologous B cells and measured IgG4 concentrations in the culture supernatants by ELISA. As shown in Fig. 3D, the concentration of IgG4 produced from B cells was much larger when B cells were cocultured with PD-1hi cTfh cells from IgG4-DS patients than when B cells were cocultured with PD-1hi cTfh cells from healthy volunteers. These results indicate that PD-1hi cTfh cells from IgG4-DS patients had a greater capacity than that of PD-1hi cTfh cells from healthy volunteers to help B cells produce IgG4. Treatment with glucocorticoids for IgG4-RD is effective and improves clinical manifestations in patients with IgG4-RD (1). We next investigated whether glucocorticoid treatment affected the proportion of PD-1hi cTfh cells. As shown in Fig. 3E and 3F, after glucocorticoid treatment, the percentage and number of PD-1hi cTfh cells were significantly decreased in accordance with reduction of serum IgG4 level. These findings indicate the clinical significance of PD-1hi cTfh cells in the production of IgG4 and disease progression in IgG4-DS
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FIGURE 2. Characteristic features of Tfh cells in tissue lesions of IgG4-DS. (A) Quantitative realtime PCR showing the expression of BCL6, CXCL13, IL-4, IL-10, and IL-21 mRNAs in Tfh cells from IgG4-DS SMGs (SMG) and tonsils (tonsil). (B) Representative histograms showing BCL6 expression in Tfh cells from IgG4-DS SMG, tonsil, and normal blood. Open histogram: blood Tfh (black), tonsillar Tfh (blue), and IgG4-DS SMG Tfh (red); closed histogram: isotype control. Mean fluorescence intensities (MFIs) of BCL6 in PD-1hiICOShi Tfh cells from IgG4-DS SMGs (SMG), tonsils (tonsil), and normal blood (blood) are shown. (C) IgG4-DS SMG Tfh (SMG) or tonsillar Tfh cells (tonsil) were cocultured with allogenic blood B (CD32CD19+) cells. Graph showing IgG1 and IgG4 concentrations (ng/ml) in the culture supernatants. Results are expressed as mean 6 SD of four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant.
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cases. Although PD-1hi cTfh cells have a lower expression level of BCL6 than do lymphoid counterparts (14) (Fig. 2B), our results presented in Fig. 3D and results of other studies show that PD-1hi cTfh cells have a capacity to help B cells produce Igs including IgG4 (12, 23). Thus, PD-1hi cTfh cells may be functionally relevant to lesional Tfh cells in IgG4-DS SMGs, which have a higher capacity to help B cells produce IgG4 (Fig. 2C). Because it was recently reported that serum level of IgG4 does not always define the activities of IgG4-RD (4), our findings indicate that the percentage of PD-1hi cTfh cells can potentially be postulated as another marker for clinical progression of IgG4-RD rather than serum IgG4. In conclusion, Tfh cells in tissue lesions of SMGs have higher functional properties of local IgG4 production and underlie the pathogenesis of IgG4-RD. To the best of our knowledge, this is the first report to show Tfh cells as a cardinal B cell helper to produce IgG4 in lesions of IgG4-RD. Moreover, we present in this article the possibility of PD-1hi cTfh cells being a clinical marker for IgG4-RD. Focusing on a modality to control abnormal Tfh cells in tissue lesions of IgG4-RD may be a viable strategy to prevent the development of IgG4-RD, and our findings thus provide an opportunity for more targeted treatment of this disease.
Disclosures The authors have no financial conflicts of interest.
References 1. Stone, J. H., Y. Zen, and V. Deshpande. 2012. IgG4-related disease. N. Engl. J. Med. 366: 539–551. 2. Yamamoto, M., H. Yajima, H. Takahashi, Y. Yokoyama, K. Ishigami, Y. Shimizu, T. Tabeya, C. Suzuki, Y. Naishiro, K. Takano, et al. 2015. Everyday clinical practice in IgG4-related dacryoadenitis and/or sialadenitis: results from the SMART database. Mod. Rheumatol. 25: 199–204. 3. Khosroshahi, A., M. N. Carruthers, V. Deshpande, S. Unizony, D. B. Bloch, and J. H. Stone. 2012. Rituximab for the treatment of IgG4-related disease: lessons from 10 consecutive patients. Medicine (Baltimore) 91: 57–66. 4. Fox, R. I., and C. M. Fox. 2015. IgG4 levels and plasmablasts as a marker for IgG4related disease (IgG4-RD). Ann. Rheum. Dis. 74: 1–3. 5. Kamisawa, T., Y. Zen, S. Pillai, and J. H. Stone. 2015. IgG4-related disease. Lancet 385: 1460–1471. 6. Wallace, Z. S., H. Mattoo, M. Carruthers, V. S. Mahajan, E. Della Torre, H. Lee, M. Kulikova, V. Deshpande, S. Pillai, and J. H. Stone. 2015. Plasmablasts as a biomarker for IgG4-related disease, independent of serum IgG4 concentrations. Ann. Rheum. Dis. 74: 190–195. 7. Okazaki, K., K. Uchida, and T. Fukui. 2008. Recent advances in autoimmune pancreatitis: concept, diagnosis, and pathogenesis. J. Gastroenterol. 43: 409–418. 8. Masaki, Y., L. Dong, N. Kurose, K. Kitagawa, Y. Morikawa, M. Yamamoto, H. Takahashi, Y. Shinomura, K. Imai, T. Saeki, et al. 2009. Proposal for a new clinical entity, IgG4-positive multiorgan lymphoproliferative syndrome: analysis of 64 cases of IgG4-related disorders. Ann. Rheum. Dis. 68: 1310–1315. 9. Frulloni, L., C. Lunardi, R. Simone, M. Dolcino, C. Scattolini, M. Falconi, L. Benini, I. Vantini, R. Corrocher, and A. Puccetti. 2009. Identification of a novel antibody associated with autoimmune pancreatitis. N. Engl. J. Med. 361: 2135–2142. 10. Crotty, S. 2014. T follicular helper cell differentiation, function, and roles in disease. Immunity 41: 529–542. 11. Zhang, Y. N., J. Song, H. Wang, H. Wang, M. Zeng, G. T. Zhai, J. Ma, Z. Y. Li, B. Liao, B. F. Wang, et al. 2016. Nasal IL-4+CXCR5+CD4+ T follicular helper cell counts correlate with local IgE production in eosinophilic nasal polyps. J. Allergy Clin. Immunol. 137: 462–473. 12. Morita, R., N. Schmitt, S. E. Bentebibel, R. Ranganathan, L. Bourdery, G. Zurawski, E. Foucat, M. Dullaers, S. Oh, N. Sabzghabaei, et al. 2011. Human blood CXCR5+ CD4+ T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 34: 108–121.
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FIGURE 3. Expansion of circulating PD-1hi Tfh cells in IgG4-DS. (A) Percentages (left panel) and absolute numbers (right panel) of PD-1hi cTfh cells in total cTfh cells from healthy volunteers (healthy) and patients with IgG4-DS, SS, and atopic asthma (asthma) are shown. (B) Percentages of PD-1hi cTfh cells in total cTfh cells from IgG4DS patients with or without multiple involvement of other organs in addition to lacrimal and/or salivary glands (comorbidities) are shown. (C) Scatterplots showing relationships of serum IgG4 (mg/dl; left panel), IgG4/IgG ratio (middle panel), and number of involved organs (right panel) with the percentage of PD-1hi cTfh cells in IgG4-DS cases. (D) PD-1hi cTfh cells from patients with IgG4-DS or healthy volunteers (healthy) were cocultured with autologous blood B (CD32CD19+) cells. Graph showing IgG4 concentrations (ng/ml) in the culture supernatants. Results are expressed as mean 6 SD of four independent experiments. (E and F) Graphs showing serum IgG4 level (mg/dl) (E) and percentage (left panel) and absolute number (right panel) of PD-1hi cTfh cells (F) before (Pre) and after (Post) glucocorticoid treatment. *p , 0.05, **p , 0.01, ****p , 0.0001. ns, not significant.
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13. Ueno, H., J. Banchereau, and C. G. Vinuesa. 2015. Pathophysiology of T follicular helper cells in humans and mice. Nat. Immunol. 16: 142–152. 14. He, J., L. M. Tsai, Y. A. Leong, X. Hu, C. S. Ma, N. Chevalier, X. Sun, K. Vandenberg, S. Rockman, Y. Ding, et al. 2013. Circulating precursor CCR7lo PD-1hi CXCR5+ CD4+ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity 39: 770–781. 15. Kamekura, R., K. Shigehara, S. Miyajima, S. Jitsukawa, K. Kawata, K. Yamashita, T. Nagaya, A. Kumagai, A. Sato, H. Matsumiya, et al. 2015. Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma. Clin. Immunol. 158: 204–211. 16. Umehara, H., K. Okazaki, Y. Masaki, M. Kawano, M. Yamamoto, T. Saeki, S. Matsui, T. Yoshino, S. Nakamura, S. Kawa, et al. 2012. Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod. Rheumatol. 22: 21–30. 17. Vitali, C., S. Bombardieri, R. Jonsson, H. M. Moutsopoulos, E. L. Alexander, S. E. Carsons, T. E. Daniels, P. C. Fox, R. I. Fox, S. S. Kassan, et al. 2002. Classification criteria for Sjo¨gren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann. Rheum. Dis. 61: 554–558. 18. The committee for The Japanese Respiratory Society guidelines for management of cough. 2006. The Japanese Respiratory Society guidelines for management of cough. Respirology 11(Suppl. 4): S135–S186.
19. Nagashima, T., S. Ichimiya, T. Kikuchi, Y. Saito, H. Matsumiya, S. Ara, S. Koshiba, J. Zhang, C. Hatate, A. Tonooka, et al. 2011. Arachidonate 5-lipoxygenase establishes adaptive humoral immunity by controlling primary B cells and their cognate T-cell help. Am. J. Pathol. 178: 222–232. 20. Maehara, T., M. Moriyama, H. Nakashima, K. Miyake, J. N. Hayashida, A. Tanaka, S. Shinozaki, Y. Kubo, and S. Nakamura. 2012. Interleukin-21 contributes to germinal centre formation and immunoglobulin G4 production in IgG4related dacryoadenitis and sialoadenitis, so-called Mikulicz’s disease. Ann. Rheum. Dis. 71: 2011–2019. 21. Baumjohann, D., T. Okada, and K. M. Ansel. 2011. Cutting edge: distinct waves of BCL6 expression during T follicular helper cell development. J. Immunol. 187: 2089–2092. 22. Akiyama, M., K. Suzuki, K. Yamaoka, H. Yasuoka, M. Takeshita, Y. Kaneko, H. Kondo, Y. Kassai, T. Miyazaki, R. Morita, et al. 2015. Number of circulating follicular helper 2 T cells correlates with IgG4 and interleukin-4 levels and plasmablast numbers in IgG4-related disease. Arthritis Rheumatol. 67: 2476–2481. 23. Akiyama, M., H. Yasuoka, K. Yamaoka, K. Suzuki, Y. Kaneko, H. Kondo, Y. Kassai, K. Koga, T. Miyazaki, R. Morita, et al. 2016. Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease. Arthritis Res. Ther. 18: 167.
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Ryuta Kamekura, Kenichi Takano, Motohisa Yamamoto, Koji Kawata, Katsunori Shigehara, Sumito Jitsukawa, Tomonori Nagaya, Fumie Ito, Akinori Sato, Noriko Ogasawara, Chieko Tsubomatsu, Hiroki Takahashi, Hiroshi Nakase, Tetsuo Himi and Shingo Ichimiya
Supplementary Material
http://www.jimmunol.org/content/suppl/2017/09/15/jimmunol.160150 7.DCSupplemental
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 15 September 2017 http://www.jimmunol.org/content/early/2017/09/15/jimmun ol.1601507
Published September 15, 2017, doi:10.4049/jimmunol.1601507
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Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease Ryuta Kamekura,*,†,1 Kenichi Takano,†,1 Motohisa Yamamoto,‡ Koji Kawata,* Katsunori Shigehara,* Sumito Jitsukawa,*,† Tomonori Nagaya,† Fumie Ito,*,† Akinori Sato,* Noriko Ogasawara,† Chieko Tsubomatsu,† Hiroki Takahashi,‡ Hiroshi Nakase,x Tetsuo Himi,† and Shingo Ichimiya*
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mmunoglobulin G4–related disease (IgG4-RD) shows a chronic fibroinflammatory condition characterized by spread to various systemic organs, resulting in dysfunctions associated with IgG4 such as dacryoadenitis and sialadenitis (also so-called Mikulicz’s disease) and type 1 autoimmune pancreatitis. As named, IgG4-RD commonly shows elevated serum IgG4 concentrations and typical pathological findings including marked infiltration of IgG4+ plasma cells, storiform fibrosis, and obliterative phlebitis in *Department of Human Immunology, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan; † Department of Otolaryngology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan; ‡Department of Rheumatology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan; and xDepartment of Gastroenterology and Hepatology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan 1
R.K. and K.T. contributed equally to this study.
ORCIDs: 0000-0001-8706-6384 (K.T.); 0000-0003-2006-1751 (M.Y.); 0000-00026688-1993 (F.I.); 0000-0003-2848-6586 (H.N.). Received for publication September 13, 2016. Accepted for publication August 28, 2017.
the involved organs (1). Treatment with glucocorticoids is effective for IgG4-RD; however, relapse frequently occurs after tapering or discontinuing administration of glucocorticoids (2). Recent studies have shown that rituximab, an antiCD20 Ab that depletes B cells, is also effective in some cases of IgG4-RD (3), suggesting that defects of humoral immunity mediated by T and B lymphocytes underlie the pathogenesis of IgG4-RD. Serum IgG4 level has been used as a diagnostic marker of IgG4-RD. However, clinical significance of serum IgG4 level as a biomarker for IgG4-RD has been debated because of high false-positive and -negative rates (4, 5) and a high relapse rate (2). Recently, Wallace et al. (6) reported that plasmablasts in peripheral blood are markedly increased in patients with active IgG4-RD, and that plasmablast count is a useful biomarker for IgG4-RD. Efforts are currently being made to find an additional clinical marker for IgG4-RD for the development of a treatment strategy. The pathogenesis of IgG4-RD also remains controversial. There are several reports showing findings consistent with an autoimmune disorder (7), an allergic disorder (8), and an infection (9) underlying IgG4-RD. Ectopic germinal centers (GCs) are frequently observed in lesions of IgG4-RD (1), indicating aberrant activation of humoral immune responses in IgG4-RD. To establish Ag-specific humoral immunity, B cells form GCs of lymphoid follicles in concert with T follicular helper (Tfh) cells, which are a class of effector helper CD4+ T cells and share CXCR5 with B cells (10). Tfh cells primarily localize and work in secondary lymphoid organs, such as the spleen, lymph nodes, and tonsils, but they are also found in peripheral blood and lesions of diseases (11). In lymphoid organs, GC-Tfh cells, which express high levels of Tfh cell–related molecules such as B cell lymphoma (BCL) 6, This work was supported by Japanese Society for the Promotion of Science Grants 15K10787 (to R.K.), 15K10818 (to K.T.), 15K20214 (to K.K.), 15K16399 (to A.S.), 15K20213 (to C.T.), and 26293370 and 16K15723 (to T.H.). Address correspondence and reprint requests to Dr. Ryuta Kamekura, Department of Human Immunology, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BCL, B cell lymphoma; cTfh, circulating Tfh; GC, germinal center; IgG4-DS, IgG4-related dacryoadenitis and sialadenitis; IgG4-RD, IgG4-related disease; PD-1, programmed death 1; SMG, submandibular gland; SS, Sjo¨gren’s syndrome; Tfh, T follicular helper. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601507
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IgG4-related disease (IgG4-RD) is a newly recognized systemic chronic fibroinflammatory disease. However, the pathogenesis of IgG4-RD remains unknown. To determine the pathophysiologic features of IgG4-RD, we examined T follicular helper (Tfh) cells in lesions and blood from patients with IgG4-RD. Patients with IgG4-related dacryoadenitis and sialadenitis (IgG4DS) showed increased infiltration of Tfh cells highly expressing programmed death 1 and ICOS in submandibular glands. Tfh cells from IgG4-DS submandibular glands had higher expression of B cell lymphoma 6 and a greater capacity to help B cells produce IgG4 than did tonsillar Tfh cells. We also found that the percentage of programmed death 1hi circulating Tfh cells in IgG4-DS patients was higher than that in healthy volunteers and was well correlated with clinical parameters. Our findings indicate that anomalous Tfh cells in tissue lesions of IgG4-RD have features distinct from those in lymphoid counterparts or blood and potentially regulate local IgG4 production in IgG4-RD. The Journal of Immunology, 2017, 199: 000–000.
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CUTTING EDGE: LESIONAL Tfh CELLS IN IgG4-RELATED DISEASE Abs For flow cytometry, the following anti-human mAbs and isotype-matched control IgG were purchased from BD Biosciences (San Jose, CA): mouse anti–CD3-allophycocyanin (UCHT1), anti–CD4-allophycocyanin-Cy7 (RPAT4), anti–CD8-PE (Leu-2a), anti–CD19-allophycocyanin-Cy7 (SJ25C1), anti– CD27-FITC (M-T271), anti–PD-1-PE (EH12.1), anti–ICOS-BV421 (DX29), and anti–BCL6-PE (K112-91) mAbs and a rat anti–CXCR5-PerCP-Cy5.5 (RF8B2) mAb. A mouse anti-human CD38-BV421 (HIT2) mAb was obtained from BioLegend (San Diego, CA). For immunofluorescence microscopy, we used mouse anti-human BCL6 (LN22), CD4 (1F6), and IgG4 (HP6025) mAbs purchased from Nichirei (Tokyo, Japan).
FACS analysis and cell sorting PBMCs were isolated from heparinized blood specimens by centrifugation over a discontinuous density gradient (Lympholyte-H; Cedarlane, Burlington, ON, Canada). Tissue samples of SMGs and tonsils were mechanically disrupted into single-lymphocyte suspensions for flow cytometry and cell sorting. Cell staining using cell surface markers was performed as previously described (19). Intracellular staining for BCL6 was performed with Transcription Factor Buffer Set (BD Biosciences) as described in the protocol of the manufacturer. Samples were then analyzed and sorted using a FACSCanto II and FACSAria II, respectively (BD Biosciences). In each experiment using FACS-sorted cells, the purity of cells reached 95% after validation with reanalysis using FACSCanto II. All data were analyzed using FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).
Quantitative real-time PCR Quantitative real-time PCR was performed using a TaqMan Gene Expression Assay kit (Life Technologies, Carlsbad, CA) with the Roche LightCycler 480 Real-Time PCR Detection System (Roche Diagnostics, Mannheim, Germany) as described in the protocol of the manufacturer. For TaqMan-based detection, the amount of GAPDH mRNA was used to standardize the amounts of target transcripts including BCL6 (Hs00153368), CXCL13 (Hs00757930), IL-4 (Hs00174122), IL-10 (Hs00961622), and IL-21 (Hs00222327). The DD cycle threshold method was used to calculate the relative mRNA expression of triplicate specimens.
Immunofluorescence microscopy
Materials and Methods Study populations Characteristics of patients with IgG4-DS (n = 42), Sjo¨gren’s syndrome (SS; n = 16), and atopic asthma (asthma; n = 20) and healthy volunteers (n = 36) are summarized in Supplemental Table I. Diagnosis of IgG4-DS was performed according to the 2011 comprehensive IgG4-RD diagnostic criteria (16). Involved organs in patients with IgG4-DS are summarized in Supplemental Table II. At the first blood sampling, none of the patients had received glucocorticoid therapy. Treatment for IgG4-DS was performed according to our protocol as previously described (2). Patients with primary SS were diagnosed in accordance with the revised European criteria (17). The diagnosis of asthma was established on the basis of the Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma 2015 and Japan Respiratory Society Guidelines (18). Subjects with asthma were diagnosed by questionnaires, physical examination, spirometry, airway reversibility and hyperreactivity tests, measurement of specific IgE, and fractional exhaled NO. No patient had a history of allergen-specific immunotherapy. Before entrance to treatment protocols, blood specimens were collected from the subjects and analyzed. None of the healthy volunteers had abnormal physical or chest x-ray findings, and results of all blood tests were negative. All subjects were nonsmokers. Written, informed consent was obtained in all cases according to the Declaration of Helsinki. All protocols were approved by the Institutional Review Board of Sapporo Medical University Hospital.
Tissue specimens Tissues of the SMG, nasal polyps, and palatine tonsils were obtained from six patients with IgG4-DS (donor age range: 52–72 y), three patients with chronic rhinosinusitis (donor age range: 43–50 y), and six patients with tonsillar hypertrophy (donor age range: 31–54 y) for diagnosis or treatment at Sapporo Medical University Hospital. Normal SMG tissues were obtained from six patients with head and neck cancer (donor age range: 54–79 y) who had undergone supraomohyoid neck dissection.
For immunostaining of tissues of SMGs, 10-mm-thick frozen sections were made with a cryostat. The tissue sections were fixed with ice-cold acetone and ethanol (1:1) for 10 min. After rinsing in PBS, the tissue sections were incubated with primary Abs at room temperature for 1 h. Subsequently, the tissue sections were incubated with Alexa 488 (green)–conjugated antirabbit or Alexa 594 (red)–conjugated anti-mouse IgG (Life Technologies) at room temperature for 1 h. Then nuclei were stained using DAPI (Dojindo Laboratories, Kumamoto, Japan) in PBS at room temperature for 15 min. Some tissue sections of SMGs were stained with H&E (Sakura Finetek Japan, Tokyo, Japan). The specimens were examined and photographed with an LSM780/ELYRAS.1 microscope (Carl Zeiss, Oberkochen, Germany).
Measurement of Igs Concentrations of IgG1 and IgG4 in cell-free culture supernatants were analyzed in triplicate with ELISAs as described in the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI). The detection limits of IgG1 and IgG4 are 3.13 and 7.8 ng/ml, respectively.
B cell coculture assays Sorted Tfh (CD3+CD4+CXCR5+PD-1hi) cell subsets from SMGs, tonsils, or blood specimens were plated with blood allogeneic or autologous B cells (CD32CD19+) from healthy volunteers in a 1:1 ratio and cocultured for 12 d in the presence of 2.5 mg/ml staphylococcal enterotoxin B (SigmaAldrich, St. Louis, MO). Supernatants were analyzed by ELISA.
Statistical analysis All data are shown as mean 6 SD. Significant differences between any two groups were determined by using Student t test or the Mann–Whitney U test. Multiple-group comparisons were analyzed with one-way ANOVA or the Kruskal–Wallis test. Correlations were determined by Pearson or Spearman correlation coefficients. The p values ,0.05 were considered significant.
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programmed death 1 (PD-1), and ICOS, help neighboring GC-B cells to undergo class-switch recombination and affinity maturation (10). In contrast, Tfh cells in peripheral blood are recognized as memory Tfh cells (12, 13). Such circulating Tfh (cTfh) cells also have an intrinsic ability to provide B cell help in vitro despite having lower BCL6 expression than that in Tfh cells residing in lymphoid tissues (12, 14). We have recently reported that cTfh cells have pathological relevance to allergic airway diseases such as allergic rhinitis and bronchial asthma, and that they are potentially related to the development of allergic airway diseases (15). In this study, we first showed that a large number of Tfh (CD3+CD4+CXCR5+) cells characterized by high expression levels of BCL6, PD-1, and ICOS had infiltrated submandibular glands (SMGs) from patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) compared with SMGs from patients with head and neck cancer as controls. Functional analyses further revealed that Tfh cells in SMGs from IgG4-DS patients had a higher capacity than tonsillar Tfh cells to help B cells produce IgG4. We also found that IgG4-DS patients showed large numbers of PD-1hi cTfh cells and that their percentage was positively correlated with serum level of IgG4, ratio of IgG4 to total IgG (IgG4/IgG ratio), and number of involved organs in IgG4-DS patients. In addition, clinical remission achieved by treatment with glucocorticoids was associated with a reduction in the number of PD-1hi cTfh cells. Collectively, our findings indicate that Tfh cells in tissue lesions of IgG4-RD have features that are distinct from those in lymphoid counterparts or blood, and that the cells potentially regulate local IgG4 production in IgG4RD. This study provides novel insights into the role of activated Tfh cells in the pathogenesis of IgG4-RD.
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Results and Discussion hi hi
Functional PD-1 ICOS Tfh cells abundantly infiltrate tissue lesions of IgG4-DS
showed abundant infiltration of CD4+BCL6+ cells in submandibular sections from patients with IgG4-DS (Fig. 1D). In addition, we found that such Tfh cells concomitantly expressed PD-1 and ICOS at high levels compared with those in control SMGs, nasal polyps, tonsils, and peripheral blood (Fig. 1E). It was noted that ICOShi Tfh cells localizing in IgG4-DS SMGs distinctively expressed PD-1 at a marked level, and that such a population of Tfh cells highly expressing PD-1 was not found in other tissues including blood (Fig. 1F). Next, we sought to characterize Tfh cells in SMGs from patients with IgG4-DS. Quantitative real-time PCR analyses of functional molecules in general Tfh cells demonstrated that Tfh cells in IgG4-DS SMGs abundantly possessed transcripts encoding BCL6, CXCL13, and IL-10, whereas the amounts of transcripts encoding IL-4 and IL-21 were comparable with those in tonsils and blood (Fig. 2A). Investigation of BCL6 expression at the protein level by intracellular flow cytometry revealed that Tfh cells in IgG4-DS SMGs indeed had a larger amount of BCL6 than did Tfh cells in tonsils and blood (Fig. 2B). Collectively, the results suggested unequivocal
FIGURE 1. Abundant infiltration of PD-1hiICOShi Tfh cells in tissue lesions of IgG4-DS. (A) Results of immunofluorescence confocal microscopy showed the expression of IgG4, CD3, and CD4 in a normal human SMG (control SMG) and an SMG from a patient with IgG4-DS SMG. Nuclei were stained using DAPI. Red (middle panel), IgG4; green, CD3; red (lower panel), CD4; blue, DAPI. Consecutive sections of normal and IgG4-DS SMG tissues were stained with H&E (upper panel). (B) Representative FACS profiles indicating CD4+ T cells (CD3+CD4+) and CD8+ T cells (CD3+CD8+). Plots were pregated on CD3+ cells and examined by the levels of CD4 and CD8. Numbers indicate percentages of cells in the gate. Percentages of CD4+ and CD8+ T cells in CD3+ cells from IgG4-DS SMG (black bars) and control SMG (white bars) are shown. Results are expressed as mean 6 SD of four independent experiments. (C) Representative FACS profiles indicating CXCR5+ cells in CD4+ T cells (CD3+CD4+). Plots were pregated on CD3+CD4+ cells and examined by the level of CXCR5. Percentages of CD3+CD4+CXCR5+ cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. (D) Results of immunofluorescence confocal microscopy showed the expression of CD4 and BCL6 in a normal human SMG (control) and an SMG from a patient with IgG4-DS (IgG4-DS). Green, CD4; red, BCL6. (E) Representative histograms showing the expression of PD-1 and ICOS in CD4+CXCR5+ cells from IgG4-DS SMG, control SMG, nasal polyp, tonsil, and normal blood. Numbers indicate percentages of cells in the gate. Percentages of PD-1hiICOShi cells in CD4+CXCR5+ cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. (F) Mean fluorescence intensities (MFIs) of PD-1 in ICOShi Tfh cells from IgG4-DS SMGs, control SMGs, nasal polyps, tonsils, and normal blood are shown. Scale bars, 20 mm. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
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Ectopic GCs are frequently observed in the lesions of IgG4-RD (1), indicating infiltration of lymphocytes and aberrant activation of humoral immune responses in IgG4-RD. Therefore, we first examined histopathological features of SMGs in IgG4-DS. As shown in Fig. 1A, dense infiltration of CD3+ CD4+ T cells, as well as IgG4+ cells, was observed in SMGs from patients with IgG4-DS compared with SMGs from controls. We also found a profile of CD4+ T cells dominantly infiltrating SMGs from patients with IgG4-DS by flow cytometry (Fig. 1B). A previous study suggested that Tfh cells, which are characterized by expression of CXCR5 and are a major source of IL-21, contribute to GC formation and IgG4 production in IgG4-RD (20). We therefore examined CD4+ CXCR5+ T (Tfh) cells in SMGs from patients with IgG4-DS. Interestingly, the majority of CD4+ T cells infiltrating IgG4DS SMGs were Tfh cells, accounting for 72.3% of CD4+ T cells (Fig. 1C). This finding was further supported by immunofluorescence labeling confocal microscopy that
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functions of Tfh cells in the pathogenesis of IgG4-DS. Further functional analyses revealed that Tfh cells in IgG4-DS SMGs had a higher capacity than that of tonsillar Tfh cells to help B cells produce IgG1 and IgG4 (Fig. 2C). Expression level of BCL6 reflects the developmental state of Tfh cells, and repeated encounters with APCs reinforce BCL6 expression in Tfh cells (21). Although the precise origin of Tfh cells residing in IgG4-DS SMGs remains unknown, our findings indicate that such Tfh cells in IgG4-DS might have many opportunities to interact with APCs and subsequently acquire specialized features to produce IgG4. The mechanisms of class switching to IgG4 remain unknown. To eliminate the effects of B cells from different donors, we performed coculture of Tfh cells from IgG4-DS SMGs or tonsils with allogenic blood B (CD32CD19+) cells from healthy volunteers. Unfortunately, we could not use naive or unswitched memory B cells because of the difficulty in accessing additional samples from patients with IgG4-DS. Therefore, this experiment will be performed in a future study to clarify the different helper activity from Tfh cells. Clinical relevance of PD-1hi cTfh cells in IgG4-DS
Blood circulation is one of the main reservoirs of functional Tfh cells (12). We therefore examined PD-1hi cTfh (CD3+ CD4+CXCR5+PD-1hi) cells in peripheral blood from patients with IgG4-DS. In general, cTfh cells include cells expressing PD-1, which represent an active form of circulating memory Tfh cells (13). We initially examined the percentage and absolute number of PD-1hi cTfh cells in total cTfh cells from patients with IgG4-DS by flow cytometry (Fig. 3A). We also analyzed cTfh cells in patients with SS and patients with asthma for comparison with those in patients with IgG4-DS. The patients with IgG4-DS showed a much larger proportion and number of PD-1hi cTfh cells in total cTfh cells than those in healthy volunteers (Fig. 3A). The patients with SS also showed a high proportion, but not number, of PD-1hi cTfh cells. In contrast, the proportion and number of PD-1hi cTfh
cells in patients with asthma were not significantly different from those in healthy volunteers. To further understand the clinical relevance of PD-1hi cTfh cells in IgG4-RD patients, we examined the percentage of PD-1hi cTfh cells in IgG4-DS patients with or without multiple involvement of other organs in addition to lacrimal and/or salivary glands (comorbidities). As shown in Fig. 3B, the percentage of PD-1hi cTfh cells was higher in IgG4-DS patients with comorbidities. We also studied the relationships between percentage of PD-1hi cTfh cells and chief clinical findings including serum IgG4 level, IgG4/IgG ratio, and number of involved organs as diagnostic parameters (Fig. 3C). Notably, the percentage of PD-1hi cTfh cells was significantly correlated with serum level of IgG4 (r = 0.5484, p = 0.0004), IgG4/IgG ratio (r = 0.4269, p = 0.0084), and number of involved organs (r = 0.6305, p , 0.0001). Similar results were obtained in a previous study by Akiyama et al. (22). To examine the functional properties of PD-1hi cTfh cells in IgG4DS patients, we cocultured PD-1hi cTfh cells with autologous B cells and measured IgG4 concentrations in the culture supernatants by ELISA. As shown in Fig. 3D, the concentration of IgG4 produced from B cells was much larger when B cells were cocultured with PD-1hi cTfh cells from IgG4-DS patients than when B cells were cocultured with PD-1hi cTfh cells from healthy volunteers. These results indicate that PD-1hi cTfh cells from IgG4-DS patients had a greater capacity than that of PD-1hi cTfh cells from healthy volunteers to help B cells produce IgG4. Treatment with glucocorticoids for IgG4-RD is effective and improves clinical manifestations in patients with IgG4-RD (1). We next investigated whether glucocorticoid treatment affected the proportion of PD-1hi cTfh cells. As shown in Fig. 3E and 3F, after glucocorticoid treatment, the percentage and number of PD-1hi cTfh cells were significantly decreased in accordance with reduction of serum IgG4 level. These findings indicate the clinical significance of PD-1hi cTfh cells in the production of IgG4 and disease progression in IgG4-DS
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FIGURE 2. Characteristic features of Tfh cells in tissue lesions of IgG4-DS. (A) Quantitative realtime PCR showing the expression of BCL6, CXCL13, IL-4, IL-10, and IL-21 mRNAs in Tfh cells from IgG4-DS SMGs (SMG) and tonsils (tonsil). (B) Representative histograms showing BCL6 expression in Tfh cells from IgG4-DS SMG, tonsil, and normal blood. Open histogram: blood Tfh (black), tonsillar Tfh (blue), and IgG4-DS SMG Tfh (red); closed histogram: isotype control. Mean fluorescence intensities (MFIs) of BCL6 in PD-1hiICOShi Tfh cells from IgG4-DS SMGs (SMG), tonsils (tonsil), and normal blood (blood) are shown. (C) IgG4-DS SMG Tfh (SMG) or tonsillar Tfh cells (tonsil) were cocultured with allogenic blood B (CD32CD19+) cells. Graph showing IgG1 and IgG4 concentrations (ng/ml) in the culture supernatants. Results are expressed as mean 6 SD of four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant.
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cases. Although PD-1hi cTfh cells have a lower expression level of BCL6 than do lymphoid counterparts (14) (Fig. 2B), our results presented in Fig. 3D and results of other studies show that PD-1hi cTfh cells have a capacity to help B cells produce Igs including IgG4 (12, 23). Thus, PD-1hi cTfh cells may be functionally relevant to lesional Tfh cells in IgG4-DS SMGs, which have a higher capacity to help B cells produce IgG4 (Fig. 2C). Because it was recently reported that serum level of IgG4 does not always define the activities of IgG4-RD (4), our findings indicate that the percentage of PD-1hi cTfh cells can potentially be postulated as another marker for clinical progression of IgG4-RD rather than serum IgG4. In conclusion, Tfh cells in tissue lesions of SMGs have higher functional properties of local IgG4 production and underlie the pathogenesis of IgG4-RD. To the best of our knowledge, this is the first report to show Tfh cells as a cardinal B cell helper to produce IgG4 in lesions of IgG4-RD. Moreover, we present in this article the possibility of PD-1hi cTfh cells being a clinical marker for IgG4-RD. Focusing on a modality to control abnormal Tfh cells in tissue lesions of IgG4-RD may be a viable strategy to prevent the development of IgG4-RD, and our findings thus provide an opportunity for more targeted treatment of this disease.
Disclosures The authors have no financial conflicts of interest.
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FIGURE 3. Expansion of circulating PD-1hi Tfh cells in IgG4-DS. (A) Percentages (left panel) and absolute numbers (right panel) of PD-1hi cTfh cells in total cTfh cells from healthy volunteers (healthy) and patients with IgG4-DS, SS, and atopic asthma (asthma) are shown. (B) Percentages of PD-1hi cTfh cells in total cTfh cells from IgG4DS patients with or without multiple involvement of other organs in addition to lacrimal and/or salivary glands (comorbidities) are shown. (C) Scatterplots showing relationships of serum IgG4 (mg/dl; left panel), IgG4/IgG ratio (middle panel), and number of involved organs (right panel) with the percentage of PD-1hi cTfh cells in IgG4-DS cases. (D) PD-1hi cTfh cells from patients with IgG4-DS or healthy volunteers (healthy) were cocultured with autologous blood B (CD32CD19+) cells. Graph showing IgG4 concentrations (ng/ml) in the culture supernatants. Results are expressed as mean 6 SD of four independent experiments. (E and F) Graphs showing serum IgG4 level (mg/dl) (E) and percentage (left panel) and absolute number (right panel) of PD-1hi cTfh cells (F) before (Pre) and after (Post) glucocorticoid treatment. *p , 0.05, **p , 0.01, ****p , 0.0001. ns, not significant.
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CUTTING EDGE: LESIONAL Tfh CELLS IN IgG4-RELATED DISEASE
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