Altered frequency of peripheral B‐cell subsets and their correlation with disease activity in patients with systemic lupus erythematosus: A comprehensive analysis

Abstract Alternations of peripheral B‐cell subsets are closely related to disease activity in systemic lupus erythematosus (SLE) and may also predict the relapse of SLE. In this study, we aimed to comprehensively analyse the frequency of peripheral B‐cell subsets, and their correlation with disease activity in patients with SLE. The results showed that for B‐cell subsets in the antigen‐independent differentiation stage, the frequency of the peripheral hematopoietic stem cell (HSC) subset in all patients with SLE was significantly higher than that of control patients. Surprisingly, several significant correlations were noted in newly diagnosed patients with SLE including a positive correlation in the frequency of the common lymphoid progenitor cell (CLP) with cholesterol serum levels. For B‐cell subsets in the antigen‐dependent differentiation stage, the frequency of naïve B‐cell (N‐B) subsets in all patients with SLE was significantly higher than that in the control patients. Moreover, the frequency of plasmablasts positively correlated with the SLEDAI score in the newly diagnosed patients. For memory B‐cell (M‐B) subtypes in the antigen‐dependent differentiation stage, the frequency of the class‐switched memory B‐cell (CSM‐B) subsets was positively correlated with the serum levels of complement C3. Notably, the frequency of the CSM‐B subset also negatively correlated with the SLEDAI score, whereas the non–class‐switched memory B‐cell (NSM‐B) subset was positively correlated with the serum levels of haemoglobin. Collectively, these findings may contribute to a better understanding of the role played by different B‐cell subsets in the pathogenesis of SLE.


| INTRODUC TI ON
Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease that affects nearly all organs, including the skin, joints, kidneys and central nervous system. 1,2 The incidence of SLE is approximately 1-10/100 000/year, its prevalence approximately 20-70/100,000, and the disease often occurs in women of childbearing age. 3 SLE is characterized by the production of large amounts of autoantibodies, forming autoantigen immune complexes that are deposited in local tissues, inducing inflammatory reactions and tissue damage. 4,5 B cells are known to play a key role in the development of SLE. 6 Moreover, the role of hyperactivated B cells in the production of autoantibodies and inflammatory cytokines is well established. 4,5 B-cell hyperproliferation and autoimmune B-cell clearance disorders lead to the production of a variety of autoantibodies, 7 causing systemic inflammation and organ damage. 8 In addition, activated B cells can also accelerate the SLE pathogenesis by secreting inflammatory cytokines such as IL-6, IL-10, IL-12 and TNF-α. 9,10 Studies have shown that the targeting of B cells may result in a good therapeutic effect in SLE, 11,12 which further proves the importance of B cells in SLE pathogenesis.
Depending on their developmental phase, B cells are divided into two stages, namely the antigen-independent differentiation and antigen-dependent differentiation stages. B-cell development is a highly regulated process that varies depending on the microenvironment. 13 Abnormalities in the development or differentiation of B cells, as well as in their activation, may lead to various immune system disorders and autoimmune diseases, such as SLE, rheumatoid arthritis and tumours. 7,[15][16][17] Notably, alternations of peripheral B-cell subsets are closely related to disease activity in SLE and may also predict patient relapse. According to CD5 expression, B cells are distinguished into B1 (CD5 + ) and B2 (CD5 -) subpopulations. 18 B1 cells exert anti-infectious effects by participating in non-specific immunity and also produce a variety of reactive autoantibodies. 19,20 B2 cells play a major role in antigen presentation, mediating specific humoral immune responses and immune regulation. In an attempt to analyse SLE pathogenesis, a previous study focused on the origins of the B1 or B2 antibody subpopulation. 21 However, neither B1 nor B2 cells account entirely for the frequency levels of the B-cell subsets; furthermore, most studies on lymphocyte subsets in the peripheral blood of patients with SLE have focused on the frequency of T-cell subsets. [22][23][24][25] In this study, we comprehensively evaluated the altered frequency of peripheral B-cell subsets, and their correlation with disease activity in patients with SLE. These findings may contribute to a better understanding of the role played by different B-cell subsets in the pathogenesis of SLE.  27 In each patient, the haemoglobin, serum albumin, serum creatinine, blood uric acid, cholesterol, serum complement C3 and C4, anti-dsDNA IgG, and SLEDAI score were determined. Table 1 summarizes patients' age, gender, positive rate anti-dsDNA IgG and anti-nuclear IgG, SLEDAI score, and therapy.

| Patients and clinical parameters
The study was approved by the ethics committee of our hospital.
Written informed consent was obtained from all patients and control patients.

| Flow cytometry analysis
Blood was collected from patients with SLE and control patients. The

| Statistical analysis
All data were analysed using SPSS statistics software (version 20.0; IBM Corp., Armonk, NY, USA) and expressed as mean ± SD or median [range] according to their distribution. Two-group comparisons were performed using unpaired two-tailed Student's t test. Multiplegroup comparisons were performed using one-way analysis of variance, followed by least significant difference or Dunnett's T3 post hoc tests. The proportions of B-cell subsets and the clinical parameters in patients with SLE were evaluated by Spearman correlation as a similarity metric and were represented as a heat map.

| Frequency of B cells in the antigenindependent differentiation stage in peripheral blood
Early B-cell differentiation in the bone marrow can be divided into five substages, HSC, CLP, Pro-B, Pre-B and Im-B. Some of these cell populations may be released into the peripheral blood and migrate to peripheral lymphoid organs. However, the frequency of these B cells in the peripheral blood of patients with SLE is not fully elucidated. We first measured the frequency of B cells in the antigenindependent differentiation stage in the peripheral blood of control patients and patients with SLE ( Figure 1A). The results showed that the frequency (percentage of total lymphocytes) of peripheral HSC subsets significantly decreased in all three patient groups: newly diagnosed, treated and total patients, compared to that in the controls (all P < 0.01) ( Figure 1B).

| Frequency of B cells in the antigen-dependent differentiation stage in peripheral blood
Im-Bs are released from the bone marrow into the peripheral blood and migrate to peripheral lymphoid organs, such as the spleen  This process, also known as antigen-dependent differentiation, involves Im-Bs, N-Bs, M-Bs and plasmablasts. Subsequently, we measured the frequency of B cells in the antigen-dependent differentiation stage in the peripheral blood of control patients and patients with SLE ( Figure 2A). Here, we observed that the frequency of the N-B subset significantly increased in patients with SLE compared to that in the control patients (for all patient groups, P < 0.05) ( Figure 2B), whereas the frequency of the M-B subset significantly decreased in all patient groups (all P < 0.001) ( Figure 2B). In addition, the frequency of the plasmablast subset significantly increased in both the newly diagnosed patients and total patient group (all P < 0.05), but not in the treated patients ( Figure 2B) compared to that in the controls. Moreover, the frequency of the plasmablast subset was significantly higher in the newly diagnosed group than in the treatment group (P < 0.01) ( Figure 2B).  Figure 3A). The results showed that the frequency of the NSM-B subset significantly decreased in all three patient groups (all P < 0.001) compared to that in the control patients ( Figure 3B).

| Frequency of M-Bs in the antigen-dependent differentiation stage in peripheral blood
Additionally, the CSM-B frequency significantly increased in the newly diagnosed patients, but not in the two other patient groups, compared to that in the control patients (P < 0.05) ( Figure 3B).

| Correlation between B-cell subsets and clinical parameters in newly diagnosed patients with SLE
The correlation between B-cell subsets and the clinical parameters in newly diagnosed patients with SLE was analysed using the Spearman method. Surprisingly, the results showed that in the antigen-independent differentiation stage, the frequency of both the HSC and

| D ISCUSS I ON
SLE is a multifactorial, autoimmune disease involving multiple organs and is characterized by immune system disorders. 28

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.