Abnormal B Cell Compartment Associated With Tfh Cells in Children With Henoch-schonlein Purpura

Ning Zhang The First A liated Hospital of Jinzhou Medical University https://orcid.org/0000-0003-1945-4734 Ge Tian First A liated Hospital of Jinzhou Medical University Yuanyuan Sun First A liated Hospital of Jinzhou Medical University Jing Pan First A liated Hospital of Jinzhou Medical University Wei Xu First A liated Hospital of Jinzhou Medical University Zhe Li (  lz2205@126.com ) First A liated Hospital of Jinzhou Medical University


Introduction
Henoch-Schonlein purpura (HSP), the most common type of childhood vasculitis, is characterized by the deposition of systemic IgA immune complexes in the walls of small vessels (1); it is estimated that 1/5000 children acquire HSP per year (2). Although considered to be a self-limiting condition, HSP is manifested by skin purpura, arthritis, abdominal pain and renal involvement; the exact pathogenesis of HSP remains unknown.
HSP is a systemic in ammatory disease that has exhibited a correlation between immune index and leucocyturia, hematuria and proteinuria. In ammatory cytokines, such as interleukin (IL)-6, IL-10 and IL-17, were reported to be involved in the pathogenesis of HSP (3)(4)(5). In addition, elevated serum IgA and IgA-related immune complexes were reported to play an essential role in the pathogenesis of HSP.
Recently, IgA-producing B cells were also found to be related to children with HSP (6). Dysregulated B cell subpopulations were found in a variety of autoimmune diseases, such as IgG4related disease and primary Sjӧgren's syndrome (7). The production of high-a nity antibodies results from the interactions between B cells and T follicular helper (Tfh) cells; an increased number of circulating Tfh cells has been correlated with the severity of autoimmune diseases (8,9).
Whether children with HSP possess abnormal B cell subsets remains unknown. Therefore, in the present study, we sought to determine differences in the population of B cells and related subsets between children with HSP and healthy control patients. Our results may provide insight into the potential role of B cell subsets and circulating Tfh cells in the pathogenesis of HSP.

Clinical Demographics
A total of 14 children diagnosed with HSP, and age-and gender-matched healthy controls (HCs) from May 2017 to March 2018 in The First A liated Hospital of Liaoning Medical University (Jinzhou, China) were enrolled in our study. The average age of all patients was 7.5 ± 2.1-years-old and 7.2 ± 1.9-years old of healthy controls. The gender ratio of HSP patients and HCs was 8/6 and 7/7 (male/female), and the average count of white blood cells was 11.5 ± 2.3 and 6.5 ± 1.8, respectively. The basic characteristics of HSP patients and HCs are present in Table I Statistical Analysis SPSS 21.0 software was used for data analysis. A Student's t test was applied to determine signi cance between two groups. Spearman's correlation coe cient was used for analyzing the correlation between variables. P < 0.05 was considered statistically signi cant.

Frequency of Expanded B cells in Children with HSP
To investigate the potential role of B cells in children with HSP, we isolated PBMCs from HSP and HC patients; the total variability of PBMCs was reported as > 95%. We analyzed the percentage of total CD3 − CD19 + B cells, as well as B-cell-related subsets, such as plasma cells (CD27 ++ CD38 ++ ), naïve B cells (IgD + CD27 − ), class-switched B cells (IgD + CD27 + ) and memory B cells (IgD − CD27 + ) (Fig. 1). As shown in Fig. 2A, the frequency of total B cells was signi cantly increased in children with HSP compared with the control group (p < 0.05); however, the percentage of plasma cells was signi cantly lower in HSP children (p < 0.05, Fig. 2B). The percentage of naïve B cells was signi cantly decreased, while that of classswitched B cells was signi cantly increased in children with HSP. There was no signi cant difference in the number of memory B cells between HSP and HC children (Fig. 2C). Collectively, we speculated that the development of abnormal B cell subsets may occur in children with HSP.
Increased Expression of CXCR5 on CD4 + T cells in Children with HSP Tfh cells, which provide specialized cognate help to B cells, were characterized as CXCR5-and PD-1positive by ow cytometry. Next, we sought to determine whether the number of peripheral Tfh cells was increased by ow cytometry (Fig. 3). We observed a decreased percentage of total CD3 + and CD4 + T cells, but not for that of CD8 + T cells in HSP children compared with the control group (Fig. 4A). Both CXCR5 expression on total CD4 + T cells and the percentage of CD4 + CXCR5 + cells were signi cantly increased in HSP patients (Fig. 4B). However, neither PD-1 expression on CD4 + CXCR5 + cells nor the percentage of CD4 + CXCR5 − cells signi cantly differed between HSP and HC patients (Fig. 4C).

The Correlation of B cell Subsets and Tfh cells in Children with HSP
We measured the percentage of B cells proportions in total 14 included patients. However, for some unexpected reasons, we did not acquire the data of Tfh cells from one patient. Therefore, the sample size of Fig. 5A-C was 13. The percentage of CD24 + + CD38 + + plasma cells from another two patients was nearly 0, therefore we excluded the data of another two patients in Fig. 5D. Correlation analysis revealed the percentage of CD4 + CXCR5 + cells to signi cantly correlate with the percentage of total CD3 − CD19 + B cells (Fig. 3A). Furthermore, CXCR5 expression on total CD4 + T cells and the percentage of CD4 + CXCR5 + cells signi cantly correlated with the percentage of naïve B cells (IgD + CD27 − ), class-switched B cells (IgD + CD27 + ) and memory B cells (IgD − CD27 + ); however, no correlation with plasma cells was observed (Fig. 5). Moreover, we did correlate B or Tfh cells with the clinical severity of HSP, but no signi cantly difference was observed (data not shown).

Discussion
Emerging evidences have shown that HSP within children is a systemic disease; however, the pathogenesis of HSP remains unknown. Functional mutations of T cells, such as abnormal cytokine secretion, were reported to be involved in the pathogenesis of HSP (11). Abnormal B cell activation with increased IgA secretion indicated that humoral immunity underlies the manifestations of HSP; however, whether abnormal B cell development and Tfh-dependent B cell responses play a role in the pathogenesis of HSP remain unknown.
In the present study, we enrolled children diagnosed with HSP, as well as HCs; the percentage of total CD3 + CD19 − B cells in each group was determined by ow cytometry. We found that the percentage of total B cells was signi cantly increased in children with HSP compared with HC patients. However, the percentage of CD27 ++ CD38 ++ plasma cells was signi cantly reduced in children with HSP.
To the best of our knowledge, no studies have been conducted to investigate the populations of total B cells and plasma cells in association with HSP. Our study is the rst to reveal an expansion of total B cells and a reduced frequency of plasma cells in children with HSP. Further study was still needed to discover the detail mechanisms.
Functionally distinct B cell subsets can be divided into different subsets by the phenotypic expression of CD27 and IgD. IgD + CD27 − B cells were de ned as naïve B cells, whereas CD27 expression by B cells has been considered as a hallmark for somatic hypermutation and memory. CD27 + memory B cells can also be divided into pre-switch (IgD + CD27 + ) and post-switch (IgD − CD27 + ) B cell subsets. We sought to determine whether differences in the B cell compartment occur in HSP children and our results showed that the percentage of naïve B cells was signi cantly reduced, while that of class-switched B cells was signi cantly increased in children with HSP. Our results indicated that the abnormal development of B cells might be related to abnormal B cell responses in children with HSP.
A large number of studies have shown that Ig production was associated with the frequency of Tfh cells in a variety of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In the current study, the percentage of CD3 + , CD4 + and CD8 + T cells was determined. In accordance with previous reports, our results showed that the frequency of CD3 + and CD4 + T cells was signi cantly decreased in children with HSP compared with HCs. CXCR5, a marker of Tfh cells, mediates the migration of Tfh cells to B cells within follicular areas containing germinal centers. We showed signi cant increases in the expression of CXCR5 on CD4 + T cells and the total frequency of CXCR5 + CD4 + Tfh cells in children with HSP compared with HC patients. PD-1, a functional marker of Tfh cells, exhibited no differences in expression between the two groups. A similar nding reported an expansion of circulating Tfh cells in children with acute HSP; a signi cant increase in CXCR5 + CD4 + cells in children with HSP and no differences in PD-1 expression between two groups were noted (12). Moreover, correlations between Tfh cells and naïve B cells, and class-switched B cell subsets, as well as memory B cells, were found in children with HSP in our study. We speculated that the HSP patients employed in our study may not exhibit an acute form of this disease as shown by the variations in their symptoms. Therefore, increased efforts should be made to classify HSP into different subtypes and the potential relationship between the frequency of CXCR5 + CD4 + Tfh cells and IgA-producing B cells should be explored.
This study has some potential limitations. The sample size of this study is small, which may lead to statistical bias. Large scale studies in HSP children are required in order to address this potential limitation.
In summary, our study showed abnormal B cell subsets and a Tfh cell-related abnormal B cell compartment in children with HSP. Our ndings may provide a new insight into the pathogenesis of children with HSP.

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