Elevated expansion of follicular helper T cells in peripheral blood from children with acute measles infection

Background Measles outbreaks have threatened the global elimination and eradication of measles in recent years. Measles virus (MeV)-specific antibodies are successful in clearing MeV infection. Follicular helper T (Tfh) cells play a crucial role in promoting antibody production. This study investigated the potential role of Tfh cells in peripheral blood mononuclear cells (PBMCs) from children with acute MeV infection. Results The frequencies of CXCR5+CD4+ Tfh, ICOShigh Tfh, and PD-1high Tfh cells in PBMCs and levels of IL-6 and IL-21 in plasma were significantly elevated in patients with acute MeV infection. Moreover, a positive correlation was discovered among the frequency of ICOShigh Tfh cells, plasma levels of IL-21 and optical density (OD) values of MeV-specific IgM antibodies in the patients with acute MeV infection. However, elevated plasma MeV-specific NAb titres were not associated with the frequency of Tfh, ICOShigh Tfh, or PD-1high Tfh cells in the patients with acute MeV infection. Conclusion These results suggest that an elevated Tfh cell frequency and associated molecules possibly play a key role in children with acute MeV infection, which contributes to the prevention and treatment of MeV infection in children.


Background
Measles, which is caused by measles virus (MeV), is a highly contagious disease characterized by fever, maculopapular rash, conjunctivitis, cough and Koplik spots, and other complications include pneumonia, encephalitis, and death [1,2]. The measles vaccine is a highly safe and effective vaccine for controlling and interrupting MeV infection; however, cases of measles have risen to high levels globally in recent years [3][4][5][6][7]. An estimated 110,000 deaths were reported in 2017, most of which were children under 5 years of age [8,9]. Measle cases rose by 300% in the first three months of 2019 in comparison with the same period in 2018 [10]. The elimination and eradication of measles remains a serious and emergent problem worldwide.
Evidence suggests that MeV-specific IgM/IgG antibody titres in serum are unexpectedly low among cohorts who had high coverage of two-dose vaccination, and low levels of MeV-specific antibodies are closely associated with outbreaks of measles infection; conversely, high levels of MeV-specific antibodies are required to achieve elimination and eradication of MeV infection [4,[11][12][13]. MeV-specific antibodies are produced by mature B cells that are helped by Th2 cells characterized by the cytokines interleukin (IL)-4, IL-10 and IL-13 [2,14]. Recent studies indicate that follicular helper T (Tfh) cells play a crucial role in promoting responses in the germinal centre (GC) by B cells that can secrete specific antibodies, and Tfh cells are characterized by expression of chemokine (CXC motif) receptor 5 (CXCR5), programmed death-1 (PD-1), and inducible costimulator (ICOS); high secretion of IL-21; and activity of B-cell lymphoma-6 (Bcl-6) as a critical transcription factor [15][16][17][18]. Peripheral ICOS high CXCR5 + CD4 + Tfh (ICOS high Tfh) and/or PD-1 high Tfh cells have been defined as circulating Tfh cells in the peripheral blood, and circulating Tfh cells are closely related to autoantibody levels in autoimmune diseases [19]. To date, there have been few studies on Tfh cells in the peripheral blood of children with acute MeV infection.
The present research explores the role of Tfh cells, associated molecules and plasma MeV-specific antibodies in peripheral blood from children with acute MeV infection. These results indicate that an elevated frequency of Tfh cells plays a crucial role in children with MeV infection, which provides new insights into the prevention and treatment of MeV infection.

Demographics of the MeV patients
Among 42 patients, 9 (21.4%) were negative, 33 (78.6%) were positive for MeV-specific IgM antibodies, and 26 (61.9%) were positive for MeV-specific IgG antibodies. Moreover, the results showed that 8 cases were associated with the Chinese vaccine strain Shanghai-191 (GenBank accession No. FJ416067) and 34 cases were positive for wild-type MeV infection. However, 18 (90%) children were positive for MeVspecific IgG antibodies in the HC group, and the results indicated that the HCs were negative for MeVspecific IgM antibodies and MeV RNA.
The mean age of the patients, including 19 females and 23 males, was 3.6 years, and the mean age of the HCs, including 9 females and 11 males, was 3.5 years. Thirty-nine patients and 20 HC children had two doses of the measles vaccine, and only 3 patients were unvaccinated. The patients with MeV infection had fever (100%), maculopapular rash (100%), cough (83.3%) and conjunctivitis (54.8%). The results are shown in Table 1.

Elevated frequencies of Tfh cells in patients with acute MeV infection
To explore the potential role of Tfh cells in the peripheral blood of the patients with acute-phase MeV infection, the frequencies of CD4 + CXCR5 + Tfh, ICOS high Tfh and PD-1 high Tfh cells in PBMCs were detected by flow cytometry (Fig. 1a). The results indicated that the frequency of CD4 + CXCR5 + Tfh cells was surprisingly increased in the patients with acute MeV infection in comparison with the HCs (Fig. 1b). In addition, the frequencies of ICOS high Tfh cells and PD-1 high Tfh cells were obviously increased in the patients with MeV infection in comparison with the HCs (Fig. 1c, d).

Correlation of plasma MeV-specific antibody levels and Tfh cell frequencies
The frequencies of CXCR5 + CD4 + Tfh and ICOS high Tfh cells but not that of PD-1 high Tfh cells were positively correlated with the optical density (OD) values of plasma MeV-specific IgM antibodies from the patients during the acute phase of MeV infection (Fig. 2a, c). Interestingly, the frequencies of Tfh cells, ICOS high Tfh cells and PD-1 high Tfh cells were significantly increased in the patients positive for MeV-specific IgM antibodies compared to those lacking specific IgM antibodies ( Fig. 2d-f). However, the frequencies of Tfh, ICOS high Tfh and PD-1 high Tfh cells were not correlated with the OD values of plasma MeV-specific IgG antibody levels (Fig. S1). Additionally, measles neutralizing antibody (NAb) titres were obviously lower in the vaccine strain (Shanghai-191) group than in the wild-type measles group, and the MeV-NAb titres were not also correlated with the frequency of Tfh, ICOS high Tfh, or PD-1 high Tfh cells (Fig. S2).

Elevated plasma IL-21 levels in patients with acute MeV infection
To investigate the role of Tfh cell-associated cytokines in the plasma, the concentrations of IL-21 and IL-6 in the plasma of the patients with acute-phase MeV infection and that of the children in the HC group were tested by ELISA. The concentrations of IL-21 and IL-6 in the plasma were remarkably  Early studies showed that Tfh cells and associated molecules facilitate the production of virus-specific antibodies to neutralize the virus by generating long-lived  [21][22][23]25]. However, limited information is available about Tfh cells in MeV infection. In this study, the frequencies of CD4 + CXCR5 + Tfh, ICOS high Tfh and PD-1 high Tfh cells in peripheral blood were significantly increased in children with acute-phase MeV infection. In addition, the Tfh cell and ICOS high Tfh cell frequencies were positively correlated with the OD values of plasma MeV-specific IgM antibodies. However, the frequencies of Tfh, ICOS high Tfh and PD-1 high Tfh cells were not related to the OD values of plasma MeV-specific IgG antibody or measles neutralizing antibody (MeV-NAb) titres. These findings suggested that elevated percentages of Tfh cells, ICOS high Tfh cells and PD-1 high Tfh cells might contribute to the induction of MeV-specific IgM antibody production during the acute phase of MeV infection. During the early phase of MeV infection, the MeV-specific IgG antibody and MeV-NAb titres were not rapidly induced by Tfh cells and their subsets. We will try to obtain follow-up serum samples to determine whether the presence of these circulating Tfh cells during the acute response correlates with better long-term antibody responses, including elevated MeVspecific IgG antibody and MeV-NAb levels, in the future, which is helpful for our research on the role of Tfh cells in MeV infection.
Recent research shows that IL-6 and IL-21 play a central role in regulating Tfh cell differentiation and function, including ICOS and PD-1 expression in Tfh cells [16,26]. IL-6 is expressed by multiple cell types, including APCs and B cells, and IL-21 is predominantly produced by Tfh cells [16,27]. IL-6 and IL-21 can drive Tfh cell differentiation by inducing expression of the transcription factor Bcl-6, and loss of IL-6 and IL-21 signalling significantly reduces the number of Tfh cells and attenuates viral clearance during chronic viral infections [27,28]. Moreover, IL-21 is important for B cell differentiation and the antibody response, and loss of IL-21 signalling produces an obvious defect in the differentiation of long-lived plasma cells and in the maintenance of specific antibody levels during virus infection [23,29,30]. In this study, the expression of IL-6 in the plasma and PBMCs was significantly elevated in the patients with acute MeV infection, which was consistent with previous studies [31,32]. Limited information is available about IL-21 levels in MeV infection. IL-21 levels were also increased in the plasma and PBMCs of the patients with acute MeV infection, and plasma IL-21 levels were closely correlated with the Tfh cell and ICOS high Tfh cell frequencies and OD values of MeV-specific IgM antibodies but not with the OD values of MeV-specific IgG antibodies. These findings indicate that MeV infection can induce the expression of IL-6 and IL-21 molecules that contribute to an increase in the number of Tfh cells and humoural immunity during the acute phase of MeV infection. Bcl-6, which is expressed in multiple cell types, including Tfh cells and B cells, is one of the major transcription factors for Tfh cell differentiation and is regulated by various molecules, including CD40-CD40 ligand and ICOS-ICOS ligand [15][16][17][18]33]. However, Bcl-6 mRNA expression was not significantly different between the patients with MeV infection and HCs. We concluded that in this study, we detected Bcl-6 mRNA expression in PBMCs but not in purified Tfh cells, which might indicate a generally negative effect on Bcl-6 mRNA expression.
Our study also had limitations. First, it included a relatively limited number of clinical cases and undetected titres of MeV-specific IgG antibodies and MeV-specific neutralizing antibodies; thus, it is impossible to extrapolate our results to the entire panel of MeV infections. Second, we studied the frequency of Tfh cells and some associated molecules in PBMCs rather than exploring the mechanism underlying Tfh cell differentiation and function.

Conclusion
The present study showed that the frequencies of CXCR5 + CD4 + Tfh cells, PD-1 high Tfh cells and ICOS high Tfh cells; levels of Tfh cell-associated molecules, including IL-21, IL-6 and Bcl-6; and plasma levels of MeVspecific IgM antibodies were significantly changed in peripheral blood from children collected during the acute phase of MeV infection, which suggested that abnormally expanded CXCR5 + CD4 + Tfh cells, ICOS high Tfh cells and PD-1 high Tfh cells with increased IL-21 and IL-6 levels might promote the production of MeVspecific IgM antibodies in children with acute MeV infection. Therefore, these results implied that aberrantly increased Tfh cell levels and associated molecules might play a crucial role in children with acute MeV infection, which would have therapeutic implications for MeV infection.

Demographics of children
The acute cases of MeV infection were confirmed by characteristic clinical symptoms (such as maculopapular rash, high fever and/or cough, and conjunctivitis) and laboratory diagnosis, including at least one positive result for MeV-specific IgM antibodies and MeV RNA [20,34]. MeV-specific IgM antibodies in plasma collected from the patients within 4 days of rash onset were detected with a commercially available enzyme-linked immune sorbent assay (ELISA) (Haitai Biotechnology, Zhuhai, China). MeV RNA was also tested with a quantitative real-time RT-PCR assay using throat, nasopharyngeal, urine and/or fluids of maculopapular rash samples [20]. Samples positive for MeV RNA were further amplified with an RT-PCR assay specific for the C terminus of the N gene of MeV [34]. The RT-PCR products purified with the Qiagen MinElute PCR Purification Kit (Qiagen, Hilden, Germany) were sequenced, and the sequences were analysed to identify the genotypes of MeV to discriminate the measles vaccine stain and wild-type virus by MEGA version 5.0 software (http://www. megasoftware.net). Forty-two children with acute MeV infection in the Department of Paediatrics and 20 healthy controls (HCs) matched by age and sex were recruited at the First People's Hospital of Huzhou, Zhejiang Province, China.

Measure of the measles neutralizing antibody (NAb) titre
Plasma was inactivated at 56°C for 30 min and diluted from 1:2 to 1:1024. In a 96-well cell plate, 25 μL of diluted serum was mixed with 25 μL of MeV solution (100 TCID 50 ) and neutralized for 1 h at 37°C. Then, 100 μL of Vero/SLAM cells (2 × 10 6 cells) were added to the plate with the serum-MeV mixture and cultured at 37°C for 7 days. The cytopathic effect (CPE) was observed and recorded in the study. The NAb titre of the serum without any cytopathic effects was determined by calculating the reciprocal of the highest dilution of the serum without any CPEs [35].

Isolation of peripheral blood mononuclear cells
Fresh peripheral blood specimens were collected from the HCs and patients with acute MeV infection. Isolation of peripheral blood mononuclear cells (PBMCs) was performed utilizing a Ficoll-Hypaque solution (CL5020, Netherlands) following the manufacturer's protocol. Next, the PBMCs were carefully washed twice with phosphate-buffered saline (PBS) and transferred into sterile tubes.

Elisa
Plasma IL-21 and IL-6 cytokine concentrations were assessed by ELISA (BioLegend, San Diego, CA) according to the manufacturer's protocols. MeV-specific IgM and MeV-specific IgG antibodies in plasma samples collected within 4 days of rash onset were detected with a commercially available ELISA (Haitai Biotechnology, Zhuhai, China) according to the manufacturer's protocol [20].

Statistical analysis
Statistical significance was determined by the Mann-Whitney U-test or one-way ANOVA. Spearman correlation coefficients were used to analyse correlations between variables. All analyses were performed with GraphPad Prism 8 software (GraphPad Software, Inc., CA). All p values< 0.05 were considered statistically significant.