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‐
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‐
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‐
18]. Peripheral ICOS
highCXCR5
+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.
Discussion
Measles vaccination is an effective measure to prevent and control MeV infection, although the variation in MeV and low levels of MeV-specific antibodies are important factors for the epidemic of MeV infection [
2,
12‐
14,
20]. In this study, there were 33 (78.6%) positive patients with MeV-specific IgM antibodies, and 26 (61.9%) patients were detected to have MeV-specific IgG antibodies among 42 patients with MeV infection. Additionally, PCR results showed that all patients were positive for MeV infection, including 8 with vaccine-associated measles and 34 with wild-type measles. Interestingly, 18 (90%) children were positive for MeV-specific IgG antibodies. These results indicated that a low positive rate for MeV-specific IgG antibodies and wild-type MeV were mainly responsible for MeV infection. Therefore, a high level of MeV-specific neutralizing antibodies, surveillance of variation in MeV and design of novel MeV vaccines are required for elimination of MeV infection.
Early studies showed that Tfh cells and associated molecules facilitate the production of virus-specific antibodies to neutralize the virus by generating long-lived memory B cells and plasma cells [
16,
21,
22]. Naive CD4
+T cells are clearly polarized to Tfh cell fates with high Bcl-6 and CXCR5 expression after lymphocytic choriomeningitis virus (LCMV) infection or influenza virus infection, which is important for viral clearance; however, Tfh cells are virus reservoirs in patients infected with human immunodeficiency virus (HIV) or individuals infected with simian immunodeficiency virus (SIV) [
16,
21‐
24]. During LCMV, HIV, or influenza virus infection, ICOS and PD-1 expression on Tfh cells is crucial for GC formation and humoural response induction [
21‐
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 MeV-specific 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‐
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-1high Tfh cells and ICOShigh Tfh cells; levels of Tfh cell-associated molecules, including IL-21, IL-6 and Bcl-6; and plasma levels of MeV-specific 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, ICOShigh Tfh cells and PD-1high Tfh cells with increased IL-21 and IL-6 levels might promote the production of MeV-specific 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.
Methods
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.
Flow cytometry
Single-cell suspensions were obtained, and surface markers were stained according to the manufacturers’ protocols for relevant fluorochrome-conjugated anti-human antibodies: anti-CD4-Pacific Blue, anti-CXCR5 (CD185)-PE-Cy7, anti-ICOS (CD278)-FITC, anti-PD-1 (CD279)-APC, and matched isotype controls (BioLegend, San Diego, CA). The samples were analysed on a BD FACSVerse™ flow cytometer (BD Biosciences, Sparks, USA), and FlowJo software (version 7.6.5) was used to analyse the flow cytometry data.
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].
Quantitative real-time PCR
Total RNA was extracted from the PBMCs of each patient and HC, and a reverse transcription reagent kit (Takara, Dalian, China) was used to synthesize cDNA. Quantitative real-time polymerase chain reaction (PCR) was performed to test the expression levels of target genes in triplicate with Takara SYBR Supermix (Takara, Dalian, China) as previously described [
36]. The sequences of the primers used were as follows: IL-21: forward, 5′-CACAGACTAACATGCCCTTCAT-3′, and reverse, 5′-GAATCTTCACTTCCGTGTGTTCT-3′;
IL-6: forward, 5′-AGACAGCCACTCACCTCTTCAG-3′, and reverse, 5′-TTCTGCCAGTGCCTCTTTGCTG-3′;
Bcl-6: forward, 5′-CATGCAGAGATGTGCCTCCACA-3′, and reverse, 5′-TCAGAGAAGCGGCAGTCACACT-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′; and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′. GAPDH was used as an internal control.
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.
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