Granulocytic myeloid-derived suppressor cells inhibit T follicular helper cells during experimental Schistosoma japonicum infection

Background CD4+ T helper (Th) cells play critical roles in both host humoral and cellular immunity against parasitic infection and in the immunopathology of schistosomiasis. T follicular helper (Tfh) cells are a specialized subset of Th cells involved in immunity against infectious diseases. However, the role of Tfh cells in schistosome infection is not fully understood. In this study, the dynamics and roles of Tfh cell regulation were examined. We demonstrated that granulocytic myeloid-derived suppressor cells (G-MDSC) can suppress the proliferation of Tfh cells. Methods The levels of Tfh cells and two other Th cells (Th1, Th2) were quantitated at different Schistosoma japonicum infection times (0,3, 5, 8, 13 weeks) using flow cytometry. The proliferation of Tfh cells stimulated by soluble egg antigen (SEA) and soluble worm antigen (SWA) in vivo and in vitro were analyzed. Tfh cells were co-cultured with MDSC to detect the proliferation of Tfh cells labelled by 5(6)-carboxyfluorescein diacetate N-succinimidyl ester. We dynamically monitored the expression of programmed cell death protein 1 (PD-1) on the surface of Tfh cells and programmed cell death ligand 1 (PD-L1) on the surface of MDSC at different infection times (0, 3, 5, 8 weeks). Naïve CD4+ T cells (in Tfh cell differentiation) were co-cultured with G-MDSC or monocytic MDSC in the presence, or in the absence, of PD-L1 blocking antibody. Results The proportion of Tfh cells among CD4+ T cells increased gradually with time of S. japonicum infection, reaching a peak at 8 weeks, after which it decreased gradually. Both SEA and SWA caused an increase in Tfh cells in vitro and in vivo. It was found that MDSC can suppress the proliferation of Tfh cells. The expression of PD-1 on Tfh cells and PD-L1 from MDSC cells increased with prolongation of the infection cycle. G-MDSC might regulate Tfh cells through the PD-1/PD-L1 pathway. Conclusions The reported study not only reveals the dynamics of Tfh cell regulation during S. japonicum infection, but also provides evidence that G-MDSC may regulate Tfh cells by PD-1/PD-L1. This study provides strong evidence for the important role of Tfh cells in the immune response to S. japonicum infection. Graphical abstract


Background
Schistosomiasis is a neglected public health problem in many developing countries, with more than 200 million people infected worldwide [1][2][3][4][5]. The mechanism of the host immune response to Schistosoma japonicum infection is complicated. However, our lack of knowledge of the details of this mechanism represents a research bottleneck for the prevention and control of schistosomiasis and for the development of vaccines against S. japonicum. Many immune cells are involved in the immune response to a S. japonicum infection, such as T helper type 1 (Th1), Th2, Th17, T follicular helper (Tfh), regulatory T (Treg) cells, dendritic cells (DCs) and myeloid-derived suppressor cells (MDSC).
Tfh cells belong to a subpopulation of CD4 + T cells that promote the generation of the germinal center and the production of antibodies by germinal center B cells. Recent evidence shows that Tfh cells may have other functions. For example, the inducible T-cell co-stimulator molecule on the Tfh cell surface can promote liver granuloma formation in mice infected with S. japonicum [6]. However, research on Tfh cells and their involvement in the immune response to S. japonicum infection is limited and its findings unclear.
A large number of cellular interactions participate in Tfh cell development. For example, DC programmed cell death ligand 1 ((PD-L1) is essential for limiting Tfh cell differentiation [7]. In influenza A virus infections, late activator antigen-presenting cells promote a Tfh response in the lymph nodes that drain the lungs [8]. Additionally, plasma cells are also reported to negatively regulate Tfh cell programming [9]. Moreover, the generation of Tfh cells is driven by macrophages in mice infected with S. japonicum [10]. However, little is known about the potential role of MDSC in the inhibition of Tfh cell development in S. japonicum infection.
In this study, C57BL/6 mice infected with S. japonicum were used as the model to analyse the dynamics and roles of Tfh cells during parasitic infection. Our findings indicate that granulocytic (G)-MDSC might regulate Tfh by programmed cell death protein 1 (PD-1) and PD-L1. The results of this study will contribute to our understanding of the mechanism of the host immune response, and provide a scientific basis for immunization and research on vaccines for schistosomiasis.

Mice and their infection with S. japonicum
Female C57BL/6 mice (aged 6-8 weeks) were purchased from SLAC Laboratory (Shanghai, China). All animal experiments were performed in accordance with the Chinese laws for animal protection and in adherence with experimental guidelines and procedures approved by the Institutional Animal Care and Use Committee. Schistosoma japonicum (Chinese mainland strain) cercariae were obtained from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. For the kinetic analysis of Tfh and MDSC cell populations, each mouse was infected with 20 cercariae of S. japonicum through the skin of the abdomen. At 0, 3, 5, 8 and 13 weeks post-infection, five mice were randomly chosen from the infected and normal control groups and sacrificed for further study.

Antigen preparation and mouse immunization
Schistosoma japonicum adult worms were obtained from infected rabbits, and soluble worm antigen (SWA) was prepared from the soluble fraction acquired from adult worms treated by ultrasound, as previously described [11]. The eggs of S. japonicum were purified from the livers of infected rabbits and used to prepare S. japonicum soluble egg antigen (SEA) [12,13]. SWA and SEA were diluted to a final concentration of 10 mg/ml in phosphate-buffered saline (PBS). C57BL/6 mice were randomly divided into three groups (control, SEA treated and SWA treated) with five mice in each group. Three independent experiments were carried out following the same methodology. SEA, SWA and PBS were emulsified in incomplete Freund's adjuvant (IFA; Sigma-Aldrich, ST. Louis, MO) [14]. Each mouse received a subcutaneous injection of 50 μg emulsified SEA, SWA or PBS at the same site in the back; booster injections were given twice at 14-day intervals. Two weeks after the final injection, Tfh levels in spleen cells were measured.

Cell culture by SEA and SWA stimulation
Splenocytes of C57BL/6 normal mice were cultured in 1 ml complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and 100 U of penicillin and streptomycin.
There were three treatment groups: the control group (splenocytes with PBS), the SEA stimulation group (splenocytes with a final concentration of 10 μg/ml SEA) and the SWA stimulation group (splenocytes with a final concentration of 10 μg/ml SWA). Triplicate wells were set up for each group, and 1 × 10 6 cells per well were cultured in 24-well plates for 72 h at 37 °C. The cells were collected and stained using flow cytometry to detect the proliferation of Tfh cells.

Flow cytometry analysis
Single-cell suspensions were prepared from the murine spleens at 0, 3, 5, 8 and 13 weeks post-infection. The splenocytes were squeezed by the plunger of a 5-ml syringe through a 70-μm cell strainer. Splenocytes were haemolysed in erythrocyte-lysing buffer and washed in PBS.
Anti-mouse PD-L1 (eBioscience, USA) blocking antibodies and isotypes were used at 5 μg/ml. Cells were cultured in triplicate in RPMI 1640 media for 5 days.

Statistical analysis
The differences between groups were assessed by oneway ANOVA followed by a least significant difference test using SPSS19.0 software. A P-value < 0.05 was considered statistically significant.

Dynamics of Th1/Th2/Tfh in mice infected with S. japonicum
Mice were infected with S. japonicum and euthanized at 0, 3, 5, 8 and 13 weeks post-infection. Splenocytes were harvested and the levels of Th1/Th2/Tfh specific phenotypic molecules were quantified with flow cytometry (Fig. 1a-c). The proportion of Th1 cells among CD4 + T cells increased gradually with the duration of S. japonicum infection, reaching a peak at 5 weeks, after which it decreased gradually (Fig. 1d). The proportion of Tfh cells among CD4 + T cells increased gradually with duration of S. japonicum infection, reaching a peak at 8 weeks, after which it decreased gradually. The proportion of Th2 cells among CD4 + T cells increased gradually with duration of S. japonicum infection.

Tfh levels stimulated by SEA and SWA
To further study the effects of two S. japonicum antigens, SEA and SWA, on Tfh cell generation, C57BL/6 mice were immunized with SWA and SEA. SWA and SEA were also used to induce CD4 + T cells to differentiate in vitro. SEA and SWA can cause an increase in Tfh cells both in vivo [F (2,12) = 57.834, P < 0.0001; Fig. 2a, b] and in vitro [F (2, 12) = 8.921, P = 0.004; Fig. 2c, d]. There was no significant difference in the proliferation of Tfh cells between the SEA and SWA treatments.

MDSC suppressed Tfh cell proliferation
In order to examine Tfh cell proliferative responses to MDSC, we examined CFSE-labelled Tfh cell proliferation by flow cytometry. CFSE-labelled Tfh cells proliferated in response to stimulation (treatment with anti-CD3 antibody combined with anti-CD28 antibody) while unstimulated Tfh cells did not (Fig. 3a, b). The presence of MDSC fully suppressed the proliferation of Tfh cells (Fig. 3c).

Similar trends observed between the expression of PD-1 on Tfh and PD-L1 on MDSC
We interrogated the mechanism of MDSC suppression of Tfh cell proliferation. First, we observed the kinetics of the expression of PD-1 on Tfh cells. Flow cytometric  analysis showed that the frequency of PD-1 expression in splenic Tfh cells increased continuously for 8 weeks postinfection compared to that before infection (Fig. 4a). As a receptor of PD-1, the combination of PD-L1 and PD1 can prevent the activation, proliferation and immune effect of T cells. We observed that the frequency of PD-L1 expression in splenic MDSC cells increased continuously for 8 weeks post-infection (Fig. 4b). The expression of PD-1 and PD-L1 increased with the prolongation of the infection cycle. There was a similar trend between the expression of PD-1 on Tfh and the expression of PD-L1 on MDSC in mice infected with S. japonicum (Fig. 4c). We speculate that these results provide evidence that MDSC might regulate Tfh via the PD-1/PD-L1 pathway.

Discussion
Many types of immune cells are involved in the host immune response to S. japonicum infection, such as Th1, Th2, Th17 [15][16][17], Tfh [18,19], Treg cells [20,21], DCs [22][23][24] and γδ T cells [25]. Tfh cells are a fairly recently identified subset of CD4 + T cells that have a distinct gene expression profile and act independently of Th1, Th2 and Th17 cell lineages [26]. There is some supporting evidence that the number of Tfh cells increases in some cases of infectious disease such as immune-active chronic hepatitis B and hepatitis C [27,28]. According to a very recent report, Tfh cells increased and promoted the formation of hepatic granulomas in mice infected with S. japonicum [6]. However, the role of Tfh cells in schistosome infections has not been fully defined. Study of the specific phenotypes and basic immunologic characteristics of Tfh cells is necessary to further elucidate and understand the development and functions of Tfh in various infectious diseases. In this study, we researched the dynamics of Tfh cell formation induced by different antigens of S. japonicum in infected mice. Moreover, we found that MDSC can suppress Tfh proliferation. Our results suggest that there are similar trends between the expression of PD-1 on Tfh and PD-L1 on MDSC. We hypothesized that MDSC might regulate the proliferation of Tfh cells through the PD-1/PD-L1 pathway.
In the present study, the proportion of Tfh cells in the spleen increased slowly for 3 weeks post-infection, then increased more rapidly from 3 to 8 weeks, reaching a peak at 8 weeks, and then decreased gradually. Meanwhile, the proportion of Th1 cells among CD4 + T cells increased gradually with duration of S. japonicum infection, reaching a peak at 5 weeks and then decreasing gradually. The proportion of Th2 cells among CD4 + T cells increased gradually with duration of S. japonicum infection. Two important antigens (SEA and SWA) are exposed to the host during S. japonicum infection [29]. They are both able to induce Th1, Th2 and Tfh cells. Therefore, we immunized mice with SEA or SWA and stimulated CD4 + T cells in vitro to confirm the above hypothesis, i.e. that MDSC might regulate the proliferation of Tfh cells through the PD-1/PD-L1 pathway. Our results showed that both SEA and SWA can induce the generation of Tfh cells. MDSC are a group of heterogeneous cells derived from bone marrow and are precursors of DCs, macrophages and/or granulocytes. MDSC have strong immunosuppressive and immunomodulatory effects and play an important role in tumour immune escape. Recent evidence suggests that MDSC also regulate immune responses in bacterial and parasitic infections, acute and chronic inflammation and autoimmune diseases [30][31][32][33][34]. Studies have shown that MDSC can interact with T cells directly or indirectly. It has been reported that MDSC can promote the differentiation of Th cell subsets by inducing arginase 1 (ARG1) and ARG2 production [35], or by upregulating nitric oxide synthase 2 [36], PD-L1 [37], IL-10 and transforming growth factor beta. It has been confirmed that cytokines secreted by MDSC can promote the differentiation of Treg [38][39][40][41][42] in hematopoietic stem cell transplantation, chronic hepatitis C infection, inflammatory bowel disease and other diseases. However, to our knowledge, whether MDSC regulates Tfh cells in schistosomiasis has not yet been reported. Our study showed that MDSC could suppress Tfh cell proliferation in vitro. Previous research suggested that MDSC could suppress immunity by PD-L1 in human cancers [43,44]. The ligand PD-L1 interacts with receptor PD-1, which transmits an inhibitory signal mediating the function of these cells [45]. Zeng et al.
[46] revealed that a PD-L1 blockade was associated with enhanced accumulation of Tfh cells, and upregulated expression of inducible T-cell co-stimulator and PD-1.
Our data showed that the expression of PD-1 on Tfh cells increased continuously for 8 weeks post-infection, as did the expression of PD-L1 on MDSC cells. There was a similar trend between the expression of PD-1 on Tfh and the expression of PD-L1 on MDSC in mice infected with S. japonicum. When G-MDSC or M-MDSC were co-cultured with naïve CD4 + T cells in the Tfh condition, the inhibition of G-MDSC on Tfh could be blocked by anti-PD-L1 blocking antibodies. M-MDSC did not significantly inhibit Tfh generation.
The combination of PD-L1 and PD1 can prevent the activation, proliferation and immune response of T cells. The data presented here strongly support the hypothesis that G-MDSC may regulate Tfh cells by PD-L1/PD-1 modulation, which lays a foundation for the important role of Tfh in the immune response to infectious diseases. Additional studies are required to provide more experimental evidence to verify and clarify the comprehensive mechanisms involved in regulation by G-MDSC. In vivo experiments are currently being planned to confirm that MDSC can regulate Tfh cells by PD-L1/PD-1 modulation.

Conclusions
Our study provides information on the kinetics of the generation of Tfh cells in S. japonicum-infected mice. In addition, our data show that, in S. japonicum infections, G-MDSC may suppress Tfh cell proliferation through the PD-1/PD-L1 pathway.