Distribution of Peripheral Memory T Follicular Helper Cells in Patients with Schistosomiasis Japonica

Background Schistosomiasis is a helminthic disease that affects more than 200 million people. An effective vaccine would be a major step towards eliminating the disease. Studies suggest that T follicular helper (Tfh) cells provide help to B cells to generate the long-term humoral immunity, which would be a crucial component of successful vaccines. Thus, understanding the biological characteristics of Tfh cells in patients with schistosomiasis, which has never been explored, is essential for vaccine design. Methodology/Principal Findings In this study, we investigated the biological characteristics of peripheral memory Tfh cells in schistosomiasis patients by flow cytometry. Our data showed that the frequencies of total and activated peripheral memory Tfh cells in patients were significantly increased during Schistosoma japonicum infection. Moreover, Tfh2 cells, which were reported to be a specific subpopulation to facilitate the generation of protective antibodies, were increased more greatly than other subpopulations of total peripheral memory Tfh cells in patients with schistosomiasis japonica. More importantly, our result showed significant correlations of the percentage of Tfh2 cells with both the frequency of plasma cells and the level of IgG antibody. In addition, our results showed that the percentage of T follicular regulatory (Tfr) cells was also increased in patients with schistosomiasis. Conclusions/Significance Our report is the first characterization of peripheral memory Tfh cells in schistosomasis patients, which not only provides potential targets to improve immune response to vaccination, but also is important for the development of vaccination strategies to control schistosomiasis.


Methodology/Principal Findings
In this study, we investigated the biological characteristics of peripheral memory Tfh cells in schistosomiasis patients by flow cytometry. Our data showed that the frequencies of total and activated peripheral memory Tfh cells in patients were significantly increased during Schistosoma japonicum infection. Moreover, Tfh2 cells, which were reported to be a specific subpopulation to facilitate the generation of protective antibodies, were increased more greatly than other subpopulations of total peripheral memory Tfh cells in patients with schistosomiasis japonica. More importantly, our result showed significant correlations of the percentage of Tfh2 cells with both the frequency of plasma cells and the level of IgG antibody. In addition, our results showed that the percentage of T follicular regulatory (Tfr) cells was also increased in patients with schistosomiasis.

Conclusions/Significance
Our report is the first characterization of peripheral memory Tfh cells in schistosomasis patients, which not only provides potential targets to improve immune response to vaccination, but also is important for the development of vaccination strategies to control schistosomiasis.

Introduction
Schistosomiasis remains a major public health problem in many developing countries. Estimates place the current number of infections at approximately 200 million people, with another 600 million considered at risk [1]. Although praziquantel remains highly effective in schistosomiasis treatment, it provides only short-term protection and does not block disease transmission or reinfection [2]. Furthermore, drug resistance and decreased susceptibility to praziquantel may occur with long-term use of the drug [3]. Thus, an effective vaccine against schistosome infection would be a major step towards eliminating this devastating and widespread tropical parasitic disease.
An effective anti-schistosome vaccine would immensely reduce the morbidity associated with schistosomiasis through induced immune responses leading to decrease in parasite load and reduced egg production [4,5]. The antibody dependent cell mediated cytotoxicity (ADCC) of effector immune cells such as eosinophils and macrophages has been suggested as one of the most important mechanisms of anti-schistosome vaccine-mediated protection [6][7][8]. Thus, the generation of long-term humoral immunity is a crucial component of successful vaccines. Interactions between T cells and B cells in germinal centers (GCs) are reported to be required for the generation of long-term humoral immunity [9]. Recent studies reveal that in GCs, a specialized subset of CD4 + T cells called T follicular helper (Tfh) cells, provide help to B cells to undergo proliferation, isotype switching and somatic hypermutation, resulting in long-lasting antibody (Ab) responses [10][11][12]. Thus, understanding the biological characteristics of Tfh cells in schistosomiasis patients is one of central issues to develop the vaccination strategies to control schistosomiasis.
In this study, we for the first time explored the characteristics of peripheral memory Tfh cells in patients with schistosomiasis japonica, which provides a better understanding of the role of Tfh cells in schistosomiasis and contributes to the development of the future vaccination strategies in schistosomiasis.

Ethics statement
Ethical clearance for this study was obtained from the Institutional Review Board of Nanjing Medical University, Nanjing, China (Permit Number: 2014NMUIEC001). The aims and objectives of the study were explained to each participant and written informed consent was obtained. All personal identifiers of the study notes and tapes were kept confidential and destroyed once the study was completed.

Patients and healthy controls
The study was conducted on a total of 100 subjects, and all subjects were from a village in Chizhou City, Anhui province. The subjects included 50 healthy adult controls, 50 patients with schistosomiasis japonica by egg detection using the Kato-Katz method with duplicate examination of 3 consecutive stool specimens obtained from each individual [13]. The healthy controls did not display a history, laboratory or clinical signs of schistosomal infection, did not suffer from coinfections with HBV or HCV, and did not use medication two weeks before blood collection.

Serology
To quantify the total serum IgG and IgE levels two commercial kit (Bethyl, Texas, USA) with established protocols from the manufacturer was used. Briefly, 96-well plates (Nunc MaxiSorp) were coated with 1 μg/well of capture antibody for IgE (catalog number A80-108A, Bethyl) or IgG (catalog number A80-104A, Bethyl) in Coating Buffer (0.05M carbonate-bicarbonate, pH 9.6) for 1 h at 25°C and blocked for 30 min with 200 μL/well Blocking Solution (50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0). Between each step, the plates were washed 5 times with Wash Solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0). The serum from each patient was diluted 1:2 or 1:20,000 for IgE or IgG in Sample/Conjugate Diluent Solution (50 mM Tris, 0.14M NaCl, 0.05% Tween 20, 1% BSA), and 100 μL/well was added to the plates. Known concentration of purified human IgE (catalog number RC80-108-6, Bethyl) or IgG (catalog number RS10-110-4, Bethyl) was added to each plate to obtain a standard curve. Serum samples and standards were incubated for 1 h at 25°C. IgE or IgG bound to the plates was detected by the addition of HRP-anti-human IgE (catalog number A80-108P, Bethyl) or HRP-anti-human IgG (catalog number A80-104P, Bethyl) at 1:75,000 or 1:200,000 dilution in Sample/Conjugate Diluent Solution, followed by the addition of Substrate Solution (catalog number E102, Bethyl). After 15 min, the reaction was stopped with 100 μL of 0.18M sulfuric acid solution. Absorbance was measured at 450 nm using an automated ELISA reader (BioTek Synergy HT, Texas). For each patient, the amount of total IgE or IgG was quantified in triplicate.

Statistical analyses
All data were analyzed using SPSS software (IBM, version 22). Significant Differences between specimens were determined by using Student's t test or Mann-Whitney U test. Correlations were determined by Spearman's ranking. The differences at p<0.05 were considered to be statistically significant.

Increased frequencies of total and activated peripheral memory Tfh cells in patients with schistosomiasis japonica
Both our previous study [14] and other literature [15] described the increased frequency of Tfh cells in mice with schistosome infection. However, whether Tfh cells increase in percentage in schistosomiasis patients remains unknown. To study the biological characteristics of Tfh cells in patients with schistosomiasis, a total of 50 patients and 50 healthy controls were recruited. There was no statistically significant difference in the distribution of age or gender between patients and healthy controls ( Table 1). The frequency of CD4 + T cells among total lymphocytes was comparable between patients and healthy controls, although the percentage of T cells was slightly lower in patients with schistosomiasis (Fig 1B and 1C). Next, we compared the frequencies of total peripheral memory Tfh cells (CXCR5 + CD45RA -CD4 + T cells) [16] and activated peripheral memory Tfh cells (PD-1 + ICOS + Tfh cells) [17,18] among CD4 + T or total peripheral memory Tfh cells in 50 patients with schistosomiasis japonica to 50 healthy controls. Results showed the increased frequencies of total and activated peripheral memory Tfh cells in patients with schistosomiasis (Fig 1A and 1D-1H). In addition, we found that almost all of the CXCR5 + CD45RA -CD4 + T cells expressed high level of GATA-3 (Fig 1I).

Tfh2 cells and Tfr cells, but not Tfh1 or Tfh17 cells, were significantly increased in patients with schistosomiasis japonica
Evidence supports that peripheral memory Tfh cells in human can be subdivided into three major subsets with distinguished biological functions according to expression of CXCR3 and CCR6, Tfh1 (CXCR3 + CCR6 -CD45RA -CXCR5 + CD3 + CD4 + cells), Tfh2 (CXCR3 -CCR6 -CD45RA -CXCR5 + CD3 + CD4 + cells), and Tfh17 (CXCR3 -CCR6 + CD45RA -CXCR5 + CD3 + CD4 + cells) [19]. We then determined the distribution of peripheral memory Tfh-cell subsets in healthy controls and schistosomiasis patients. Results showed that Tfh2 cells were a predominant subset of peripheral memory Tfh cells, and accounted for more than 50% of total peripheral memory Tfh cells in patients with schistosomiasis (Fig 2A and 2B). In addition, the percentage of total Th2 cells, which include Tfh2 cells, was greater in schistosomiasis patients than those in healthy controls (Fig 2C). Furthermore, we found that the percentage of Tfh2 cells, rather than that of Tfh17 or Tfh1, is significantly increased in schistosomiasis patients (Fig 2D-2G).
Given that Tfr cells were identified as a Treg cell subset specialized for suppressing B and Tfh cells [20][21][22], we next investigated the biological characteristics of circulating Tfr cells in schistosomiasis patients. Results showed that most of circulating Tfr are CD45RAcells (Fig 2H), which is consistent with the previous observation that circulating Tfr cells have memory-like properties [22]. Furthermore, we found that the percentage of circulating Tfr cells within CD4 + T cells (Fig 2I and 2J) or total Tfh cells (Fig 2K) was significantly increased in schistosomiasis patients.

Accumulation of Tfh2 cells was associated with increased frequency of plasma cells in patients with schistosomiasis japonica
Given the roles of Tfh cells in providing help to B cells, we next characterized the frequencies of different subsets of B cells by flow cytometry analysis. As shown in Fig 3, the percentages of CD27 + CD19 + CD3 -CD4memory B cells [23][24][25], CD86 + CD19 + CD3 -CD4activated B cells [26][27][28], and CD38 ++ CD19 + CD3 -CD4plasma cells [29][30][31] in schistosomiasis patients were significantly greater than those in the HCs, although the percentage of total B cells was slightly decreased in patients with schistosomiasis. In contrast, the percentage of CD27 -CD19 +-CD3 -CD4naïve B cells [32] in patients was less than that in the HCs (Fig 3C). Furthermore, the percentage of Tfh2 cells was moderately correlated with the percentage of plasma cells (r s = .362, p = .01) in schistosomiasis patients, and its correlation with the percentage of activated B   211, p = .141) followed the same trend but it did not reach statistical significance (Fig 3I and 3J).

Accumulation of Tfh2 cells was associated with increased level of IgG antibody in patients with schistosomiasis japonica
Given that Tfh2 cells are efficient at promoting IgG and IgE secretion [33], we next determined whether the frequency of Tfh2 cells was associated with the levels of total IgG and IgE antibodies in schistosomiasis patients. Results showed the increased concentrations of total IgG [HC vs. Sj: mean = 359.7, 95% confidence interval (95% CI) = 343.6-375.7 vs. mean = 417.9, 95% CI = 398.2-437.5; p<0.001] and IgE antibodies (HC vs. Sj: mean = 10.6, 95% CI = 9.5-11.7 vs. mean = 38.6, 95%CI = 30.5-46.7; p<0.001) in schistosomiasis patients (Fig 4A and 4C). Furthermore, a striking correlation between the percentage of Tfh2 cells and the level of total IgG (r s = .425, p = .002) was observed in schistosomiasis patients (Fig 4B). Although there was a tendency of correlation between the percentage of Tfh2 and the level of total IgE (r s = .173, p = .229) in schistosomiasis patients, it did not reach statistical significance (Fig 4D).

Discussion
Prevention and control of schistosomiasis demands an effective vaccine. T follicular helper cells have a pivotal role in the generation of the long-term humoral immunity and are proved to be one of crucial contributors of successful vaccines. However, the lack of knowledge about Tfh cells in schistosomiasis patients limits the ability to develop successful anti-schistosome vaccinations. Here, we characterized the distribution of peripheral memory Tfh cells in patients with schistosomiasis japonica. Our study significantly extends our understanding of Tfh cells in patients with schistosomiasis, which is helpful for vaccine design for the prevention of schistosome infection.
Although the phenotypes of Bona fide Tfh cells in GCs are easy to be analyzed by flow cytometry, it is not only difficult to get lymph nodes from schistosomiasis patients only, but from humans in general. Fortunately, studies in humans showed that peripheral memory Tfh cells share functional properties with bona fide Tfh cells in secondary lymphoid organs [17,18,[33][34][35][36], indicating the analysis of peripheral memory Tfh cells by flow cytometry is an alternative approach to study the biological characteristics of bona fide Tfh cells in human. Here, for the first time we revealed that the percentages of total and activated peripheral memory Tfh cells were significantly increased in schistosomiasis patients. These findings are in accordance with our previous observation that Tfh cells are substantially increased in schistosome-infected mice [14].
Human peripheral memory Tfh cells can be divided into three major subsets with distinguished functions according to the analysis of CXCR3 and CCR6 expression, including in CXCR3 + CCR6 -Tfh1 cells, CXCR3 -CCR6 -Tfh2 cells and CXCR3 -CCR6 + Tfh17 cells [19].
CR45RA -CXCR5 + CD4 + T cells; (C) Shown is the distribution of total Th1 (CXCR3 + CCR6 -CD4 + T cells, red), Th2 (CXCR3 -CCR6 -CD4 + T cells, purple), Th17 (CXCR3 -CCR6 + CD4 + T cells, green), and the other CD4 + T subsets (black) within total CD4 + T cells in patients with schistosomiasis japonica. Areas represent the means of percentages of Th1, Th2, Th17 or the other CD4 + T subsets with CD4 + T cells;  Results from staphylococcal enterotoxin B in vitro coculture experiment suggested that human blood Tfh2 and Tfh17,but not Tfh1, cells can help naive B cells to produce immunoglobulins via producing interleukin-21 [33]. More specifically, it suggests that Tfh2 cells are considered to be efficient at promoting IgG and IgE secretion, whereas Tfh17 cells promote IgG and IgA secretion [33]. Our data showed that the significant increase of Tfh2 cells is a major contributor to the increased frequency of total peripheral memory Tfh cells in patients with schistosomiasis japonica. These findings nicely connect to our observation that the levels of memory B cells, activated B cells, plasma cells as well as total IgG and IgE responses were considerably increased in patients with schistosomiasis. More importantly, both IgG and IgE antibodies have been reported to be essential components of protective immunity, and involved in the ADCC of eosinophils and macrophages against schistosome larvae [37][38][39], which is considered as one of the most important means of anti-schistosome vaccine-mediated protection [6][7][8]. Thus, Tfh2 cells, a predominant subset of peripheral memory Tfh cells in schistosomiasis patients, might be considered as a potential target to improve IgG and IgE responses to vaccination. However, Tfh2 cells secrete Th2 cytokines, i.e., IL-4, IL-5, and IL-13 [33]. Given that the prolonged excessive production of Th2 cytokines contributes to the development of hepatic fibrosis and chronic morbidity in schistosomiasis [40] and the progression of Th2-mediated pathology in some diseases, such as asthma and other infectious diseases caused by extracellular parasites [41], it is very important for us to consider adverse effect of anti-schistosome vaccines on triggering Th2-mediated inflammation responses, particularly on liver pathology in patients with a history of infection with schistosoma japonica, when we manipulate Tfh2 cells to enhance IgG and IgE responses to vaccination. Strikingly, we found that the frequency of circulating Tfr cells, which play a crucial role in GC responses by limiting Tfh and GC B cell numbers as well as plasma cells differentiation [20], was significantly increased in schistosomiasis patients.
In summary, our study for the first time described the distribution of peripheral memory Tfh cells, circulating Tfr cells, and B cells in patients with schistosomiasis japonica, which provides us a better understanding of the biological characteristics of these cells in patients with schistosomiasis.