Variation in IL-21-secreting circulating follicular helper T cells in Kawasaki disease

Kawasaki disease (KD) is an acute febrile vasculitis that was first reported in 1967 [1]. KD most commonly occurs in infants and children under five years of age and is characterized initially by high fever, mucocutaneous inflammation, edema of the extremities, polymorphous rash, and cervical lymphadenopathy (≥1.5 cm diameter) [2]. The exact pathogenesis of Kawasaki disease remains unknown. Generally, it is believed that KD is secondary to infectious agents, triggering an aberrant immune response in genetically susceptible individuals [3, 4]. The treatment efficacy of intravenous immunoglobulin (IVIG) administered during the acute phase of the disease further confirms the fundamental involvement of immune response dysregulation in the pathogenesis of KD. Therefore, the understanding of the factors that regulate the abnormal immune response in KD is important for developing strategies for the management of KD patients.

Both innate and adaptive immune systems are involved in the pathogenesis of KD. The innate immune system presents high numbers of activated circulating neutrophils and elevated levels of serum cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNFα) [5]. The reduction in the numbers of neutrophils and cytokines levels following IVIG administration [6] is regarded as a consequence of the downregulation of the nuclear factor kappa B (NF-κB) signaling pathway in monocytes/macrophages [7]. Although T cells have traditionally been considered to play essential roles in adaptive immune responses, the alterations in the percentage of T cells in KD are a subject of controversy and different investigators have reported conflicting results [8‐10]. It has been concluded that the variation in T cells could be further investigated based on of their subpopulations and cytokine levels [5]. Current studies that examined the variation of T-cell subpopulations with regard to KD have generally investigated T helper cells [11, 12], regulatory T cells [13], and cytotoxic T cells [14]; however, the role of follicular helper T (Tfh) cells in KD remains to be elucidated. B cells are also important participants in the adaptive immune responses. In acute phase of KD, studies about B cells found increased CD19⁺ cells [15] and elevated levels of serum immunoglobulins (IgA, IgG, IgM and IgE) [16]. Accordingly, it can be speculated that Tfh cells may be involved in the pathogenesis of KD due to their ability in homing B cell to the germinal centers (GC), regulating GC positive selection, and inducing B cell differentiation to plasma cells and memory B cells via the secretion of IL-21 [17‐19]. However, suppressed plasmablast responses [20] and reduced IgA-expressing B cells [21] in KD were also reported. Hence, the studies conducted on Tfh cells are important in clarifying the role of B cells in KD, since Tfh cells can aid the function of B cells.

Tfh cells were initially identified in the GC of secondary lymphoid tissues and have been shown to express the CXC-chemokine receptor 5 (CXCR5). The percentage of Tfh cells can be detected by the expression of inducible co-stimulator (ICOS) and programmed cell death protein 1 (PD-1), two CD28 superfamily molecules [22, 23]. ICOS delivers positive signals to CD4⁺ T cells by interacting with dendritic cells and B cells that express an ICOS ligand, which is involved in Tfh cell differentiation [24, 25]. In contrast to ICOS, the inhibitory signals [26, 27] that block the interactions between PD-1 and the ligands PD-L1 and/or PD-L2 have been shown to increase the differentiation of Tfh cells [28, 29]. The assessment of GC Tfh cells in patients, particularly in children, is subject to huge limitations, due to poor access to lymphoid organ samples. Despite these disadvantages, the establishment of circulating Tfh (cTfh) cells [26] from blood samples offers a surrogate strategy to analyze Tfh responses. Circulating Tfh cells are believed to represent a memory compartment of GC Tfh cells that express B-cell lymphoma 6 protein (Bcl-6), which is downregulated when the cells enter the circulation [30]. Circulating Tfh cells share the phenotype and functional properties of GC Tfh cells [31, 32]. Current understanding of cTfh cells suggests that they can be classified into distinct subsets according to the differential expression of ICOS and PD-1. Typically, they were divided into one activated subset (ICOS⁺PD1⁺⁺) and two quiescent subsets (ICOS⁻PD1⁻ and ICOS⁻PD1⁺) [33]. The majority of ICOS⁺PD1⁺⁺ cTfh cells express Ki67, which is a cellular marker for proliferation. On the contrary, the two quiescent subsets lack the expression of Ki67 [34]. Furthermore, in comparison, ICOS⁺PD1⁺⁺ cTfh cells display the most efficient capacity in helping naïve or memory B cells. Thus, investigation on the variation in cTfh-cell subsets would be beneficial for better understanding the level of their activation. IL-21 is recognized as a hallmark for cTfh cells [33] and belongs to the type I cytokine family. This cytokine is a highly potent stimulator of plasma cell proliferation and differentiation [35]. IL-21 is also believed to be an autocrine growth factor for Tfh cells [36]. However, the role of IL-21 in cTfh cells remains to be fully elucidated.

T cells comprise a heterogeneous population; therefore, in the present study, we aimed to analyze the distinct subsets of circulating Tfh cells that were defined by characteristic Tfh markers and serum IL-21 levels. The examination of their expression levels and the correlation among the subgroups can be used to further clarify the immunopathogenesis of KD.

The roles of circulating Tfh cells in different disease models have already been widely investigated. A previous study conducted on systemic lupus erythematosus indicated that PD-1 expression on cTfh cells contributed to the regulation of germinal center B-cell function and humoral response [38]. Szabo et al. reported that ICOS⁺PD-1⁺ cTfh cells and IL-21-producing CD4⁺CXCR5⁺PD-1⁺ T cells were significantly increased in Sjögren’s syndrome subjects [39]. Our previous study in children indicated that the percentage of cTfh cells correlated with the pathogenesis and progression of Henoch–Schönlein purpura [40]. In the present study, we found that although the overall percentage of cTfh cells remained stable, marked variations were noted in the percentages of cTfh-cell subsets and the levels of cytokines. In the AP, the percentages of ICOS^highPD-1^high, ICOS⁺PD-1⁺, ICOS⁻PD-1⁺, and CD45RA⁻IL-21⁺ cTfh cells and serum IL-21 levels were significantly elevated. It is apparent that aberrant distribution of cTfh cells is ubiquitous in rheumatic diseases, although the precise mechanism remains unclear. Based on our data, ICOS^highPD-1^high, ICOS⁺PD-1⁺, ICOS⁻PD-1⁺ cTfh cells correlated positively with CRP and ESR, which suggested that these three subsets contribute to innate immune responses. Correlation analysis with IL-21 levels implied that ICOS^highPD-1^high, ICOS⁺PD-1⁺ and CD45RA⁻IL-21⁺ cTfh cells affected KD development via IL-21 secretion. It seems difficult to explain that although the clinical symptoms had been improved in the RP, the percentages of the ICOS^highPD-1^high and ICOS⁺PD-1⁺, and ICOS⁺PD-1⁻ cTfh cells were further increased. However, among these subsets, ICOS^highPD-1^high population may be a representative subset of cTfh cells. Not only because ICOS^highPD-1^high population can be regarded as a subpopulation of ICOS⁺PD1⁺⁺ cTfh cells, which is an activated subset, but also because this phenotype is similar to that of GC Tfh cells, which are ICOS⁺⁺PD-1⁺⁺ [35]. In other words, the increase of ICOS^highPD-1^high cTfh cells likely represents the activation of cTfh cells. Hence, our results suggested that cTfh cells were activated in the AP, and they were persistently activated in the RP. Although the function of these subsets cannot be clarified in the present analysis, the absence of a correlation with IL-21 levels despite the increased percentages of these subsets, indicates that these cells may not secrete IL-21 efficiently in RP [34, 41, 42]. Furthermore, Franco et al. [43] indicated that the expansion of circulating central memory T cells but not effector memory T cells could be detected even at 1 to 3 months following the onset of the disease. This expansion aims to protect patients against future exposure to the stimuli of KD. Whether these further increased subsets in RP are circulating central memory cells remains to be clarified.

Since its discovery in 2000, IL-21 has been shown to perform antitumor and antiviral functions and to participate in the development of autoimmune diseases via the Janus kinase (JAK)-signal transducer, the activator of transcription (STAT), the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K)-AKT signaling pathways [44]; however, the mechanism underlying the role in KD is unknown. Several studies have been conducted on the role of IL-21 in KD. One study in a Korean cohort demonstrated that IL-21 represents a sensitive and specific biomarker of KD [45]. In accordance with this report, we found elevated serum IL-21 levels in the AP, while the corresponding levels were reduced in the RP. However, Engelberg et al. reported that IL-21 levels were also elevated in febrile controls [46]. Therefore, serum IL-21 levels appear to be a sensitive, but not specific, indicator of active KD. Additionally, serum IL-21 levels may be regulated by IgG levels [45]. In the present study, we observed lower levels of serum IgG and higher expression levels of IL-21 in the AP, while in the RP the IgG levels were shown to increase [47] and IL-21 levels decreased. Nevertheless, further analysis indicated no correlation between serum levels of IL-21 and IgG in the AP. It can be speculated that this finding may have been produced by the increase in the normal range of IgG with age. Furthermore, IL-21 is not exclusively expressed in cTfh cells, but also in Th17 and NKT cells [48, 49]. IL-21 was further found to be suppressed by activated plasma cells [50]. Thus, in addition to cTfh cells, multiple factors, such as Th17, NKT and plasma cells, might affect serum IL-21 levels.

The relationship between IL-21 expression and Tfh cell differentiation remains controversial. It has been postulated that IL-21 contributes to the efficient development of Tfh cells by upregulating BCL-6 and MAF [51, 52], while other studies have indicated that Tfh cells can be generated without IL-21 expression [53]. Alternatively, it has been shown that IL-21 alone is insufficient to enhance the expression of CXCR5 [54]. Thus, it can be hypothesized that IL-21 acts as a cooperator, rather than a conductor of Tfh cell differentiation, which is a complex process regulated by IL-21, IL-12, IL-6, BCL-6, and ICOS [35, 54]. In the present study, we investigated the percentage of total cTfh cells and the serum IL-21 levels in KD patients during different phases of the disease progression. It is interesting to note, that no significant variation was noted in the percentage of CXCR5 cells regardless of the levels of IL-21, indicating that no definite correlation between IL-21 levels and cTfh cell differentiation in KD.

Unfortunately, the correlations between coronary artery lesions and cTfh cells were not analyzed in the present study. However, a recent study revealed associations of coronary artery lesions with a number of clinical characteristic, particularly CRP and ESR [55]. It was noted that KD patients with CRP levels higher than 30 mg/L and ESR higher than 40 mm/h exhibited a higher incidence of coronary artery lesion. Moreover, the percentage of ICOS⁻PD-1⁺ cTfh cells correlated negatively with ESR and CRP, indicating the possibility of an increased percentage of ICOS⁻PD-1⁺cTfh cells to act as a protective barrier against coronary artery lesions. This hypothesis may provide a new therapeutic approach in preventing coronary artery lesions. According to our clinical observations, coronary artery lesions were present in the patients without significant elevation of CRP and ESR. In addition, not all of the patients with high CRP and ESR possessed coronary artery lesions. It can be speculated that the pathogenesis of coronary artery lesions in KD is diverse, possibly due to epigenetic effects [56] and genetic polymorphisms [57]. Therefore, investigation of the association between cTfh cells and the development of coronary lesions are required. Besides, another limitation of the present study shoud be mentioned. The exact function and mechanism underlying the further increase in the cTfh cell population during the RP remain to be elucidated. Focus will be given on these issues in the subsequent studies.

In summary, significant variations were identified in the percentages of cTfh-cell subsets and the level of serum IL-21 in Kawasaki disease. Our results indicated that IL-21-secreting cTfh cells were essential for both the acute and remission phase of KD. It also can be concluded that cTfh cells were activated in the acute phase and persistently activated in remission phase. To the best of our knowledge, this is the first investigation of serum IL-21 levels in terms of the distribution of circulating cTfh cell subsets in KD. Our data provide further understanding of the immune responses of KD and novel insights in the pathogenesis of this disease.