Aberrant expression of PD-1 on B cells and their subpopulations in systemic lupus erythematosus and association with clinical parameters

Background: The binding of programmed death 1 (PD-1) with its ligands inhibits the T cell activation and proliferation. But role of the PD-1 pathway on B cells is unclear. In present study, we aimed to evaluate the expression of PD-1 on B cells and their subpopulations and association with clinical parameters in systemic lupus erythematosus (SLE). Results: The frequency of B cells increased significantly in patients with active SLE compared with healthy controls and patients with inactive SLE.The proportions of CD19+ IgD- CD27- cells andplasmablast cell among total B cells were significantly higher in patients with SLE compared with controls. The percentage of PD-1+ B cells was higher in patients with in active SLE than in healthy controls. The proportion of PD-1+ B cells was correlated with lupus nephritis, complement components, IgG, SLE Disease Activity Index, and autoantibodies. PD-1+ B cells from SLE showed a high proliferative response. The levels of IgG and anti-dsDNA secreted by PD-1+ B cells from SLE patients was higher after 7 days compared with that by PD-1- B cells from patients with SLE and healthy controls. Conclusions: The expression of PD-1 on B cells and their subpopulations was aberrant and was associated with clinical parameters in SLE. B cells SLE, cells B


Background
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease triggered by the breakdown of self and non-self discrimination by the immune system and the resultant immunological abnormalities and excessive inflammatory reactions in a wide range of organs 1 . The etiology and pathogenesis of SLE are not fully understood. Nevertheless, it is well known that T and B cells are involved in the development of SLE [2][3][4][5] .
Recent studies demonstrated that programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, are involved in the process of T-cell activation, tolerance and immune-mediated tissue damage [6][7][8] . In addition, increasing evidence indicates that the PD-1/PD-L1/2 axis is crucial in preventing autoimmune disorders [9][10][11] . Experimental models of autoimmunity demonstrated that the altered function of PD-1 and its ligands is associated with several human autoimmune conditions 12-14 . Multiple studies have implicated the PD-1/PD-L1 pathway in immune system homeostasis and a variety of autoimmune diseases [15][16][17] . Blockade with anti-PD-L1 or anti-PD-1 in mice models of SLE has been shown to ameliorate lupus-like symptoms by decreasing the inhibition of T cells [17][18][19] . PD-L1-Ig treatment in New Zealand F1 mice significantly delayed the onset of proteinuria and prolonged their lifetime 20 . Nevertheless, the expression of PD-1/PD-L1 in SLE still remains unclear.
It has been reported that the proportion of IgDCD27 + class-switched memory B cells were found to be significantly high in patients with SLE, antigen-experienced, and resistant to immunosuppressive therapy 2,21 . Of note, the peripheral memory B-cell receptor (BCR) repertoire in SLE is formed by abnormal selection, which deteriorates somatic hypermutation and increases receptor editing 22 .
These memory B cells have lower activation thresholds, leading to a higher risk of autoimmunity.
Moreover, these cells can be rapidly activated in a non-antigen-specific way by linking with Toll-like receptor agonists, B-cell-activating factor, or a combination of cytokines 23 . Another memory B-cell subset particularly high in patients with SLE has been identified and is composed of B cells that do not express CD27 24 . Although IgDCD27 B cells expressing mutated BCRs have been detected in the tonsils and peripheral blood of healthy donors 25, 26 , a large number of IgDCD27 memory B cells were found in the peripheral blood of patients with SLE, and the highest levels were associated with high disease activity and active renal disease 27 .
Nevertheless, the relation between PD-1/PD-L1 and B cells in SLE is still not fully understood. Therefore, the present study aimed to explore the importance of the expression of PD-1 on B cells in SLE.

Methods
Patients SLE was diagnosed according to the 1997 American College of Rheumatology revised criteria 28 . A total of 74 patients with SLE and 54 healthy controls (all of Asian origin) were enrolled at the Department of Rheumatology of the First Affiliated Hospital of Bengbu Medical College, China. Data including age, gender, blood cell counts, 24-h proteinuria excretion, anti-dsDNA antibody, antinucleosome antibody, anti-Smith antibody (anti-Sm antibody), anti-Sjogren syndrome A antibody (anti-SSA antibody), anti-Sjogren syndrome B antibody (anti-SSB) antibody, complement component 3 (C3), complement component 4 (C4), IgG, IgM, IgA, and erythrocyte sedimentation rate (ESR) were collected from the medical records. The disease activity was scored using the SLE Disease Activity Index (SLEDAI) and classified as inactive SLE (SLEDAI <10) and active SLE (SLEDAI ≥10).
Enzyme linked immunosorbent assay (ELISA) The culture medium of the isolatedPD-1 + or PD-1 -B cells was changed every 2 days, and the anti-dsDNA and IgG levels in the supernatant were tested at 1, 3, 5, and 7 days by ELISA (Biorbyt, San Francisco, CA, USA).

Statistical analysis
All data were presented as mean ± standard deviation. The significance of the differences between groups was analyzed using one-way analysis of variance, and that between two groups was evaluated using the two-tailed Student t test. The Spearman correlation coefficient or Pearson correlation coefficient with two-tailed P value was used to analyze correlations. A P value <0.05 was considered statistically significant. All data were analyzed using SPSS 16.0 (IBM, Armonk, NY, USA). Table 1 presents the characteristics of the patients. Compared with the healthy controls, the CD19 + B-cell population was significantly higher in patients with SLE (P<0.05). The ratio of B cells was slightly higher in active SLE patients than in inactive SLE patients, but showed no statistical significance ( Fig 1A- (Table 3).

Differential expression of PD-1 on B cells
Previous studies indicated that PD-1 is an important co-inhibitory receptor in SLE. Therefore the expression of PD-1 on B cells was determined. As indicated in Fig. 2A-B, the percentage of CD19 + PD-1 + cells (PD-1 + B cells) was higher in patients with active SLE than in patients with inactive SLE and healthy controls. The percentage of CD19 + PD-1 + cells (PD-1 + B cells) was higher in patients with inactive SLE than in healthy controls. The expression levels of PD-1 on plasmablast cells, CD19 + IgD CD27 + (switched memory) cells, CD19 + IgD + CD27 + (non-switched memory) cells, CD19 + IgD CD27 cells, and naïve cells were higher in patients with SLE ( Fig. 2C-D). These findings indicated that the levels of PD-1 on B cells and their subpopulations were obviously higher in patients with SLE.
The correlation of PD-1-positive B cells with clinical data was assessed in patients with SLE. The percentage of PD-1 + B cells was found to be positively correlated with SLEDAI (Fig. 2E). Moreover, the levels of PD-1 + B cells were positively correlated with 24-h urinary protein and serum levels of IgG and inversely with IgM (Fig. 2E). The frequency of PD-1 + B cells was also higher in patients with anti-dsDNA (+) Abs (P = 0.040), anti-histone (+) Abs (P = 0.025), and anti-SSA52 (+) Abs (P = 0.048) Table 4). The frequency of PD-1 + B cells was significantly higher in patients with lupus nephritis (P <0.0001) and oral ulcer (P = 0.05) ( Table 4). No significant differences were observed between hematological manifestations, arthritis, and serositis. The association of PD-1 + in B-cell subsets with clinical manifestations and auto-antibodies in SLE was found in patients with SLE (Table 5). The percentage of PD-1 + PC B cells was positively associated with SLEDAI, 24-h urinary protein, and the levels of IgG (Fig. 2F). The percentage of PD-1 + SM B cells was positively associated with SLEDAI and 24-h urinary protein. The percentage of PD-1 + NSM B cells was positively associated with the levels of IgG. The percentage of PD-1 + N B cells was positively associated with the levels of IgG. These findings indicated that PD-1 + B cells seemed to be associated with disease activity in SLE.

Function of PD-1 + B cells in vitro
The isolated B cells (PD-1 + or PD-1 -B cells) were significantly stimulated with CpG DNA (Fig. 3A). PD-1 + B cells from patients with SLE obviously augmented after 7 days compared with PD-1B cells from patients with SLE and PD-1 + or PD-1 -B cells from healthy controls in response to CpG DNA stimulation (Fig. 3B). The levels of anti-dsDNA secreted from PD-1 + or PD-1 -B cells had no statistical significance at 1, 3,and 5 days among patients with SLE and controls (Fig. 3C), The levels of anti-dsDNA produced by PD-1 + B cells from SLE patients at 7 day were significantly higher compared with PD-1 B cells from patients with SLE or PD-1 -B cells from healthy controls (P <0.01, P <0.001,respectively, Fig. 3C). Furthermore, the amount of IgG secreted by PD-1 + B cells from patients with SLE was higher after 7 days compared with that by PD-1 B cells from patients with SLE and PD-1 + or PD-1 -B cells from healthy controls (P = 0.0261; Fig. 3D). These results showed that the abilities to proliferate and secrete IgG in PD-1 + B cells from SLE patients were high.

Discussion
The present study showed that the frequency of CD19 + B cells and the SM and DN subsets were In summary, this study showed that B cells and their subgroups were abnormal in patients with SLE.
The expression of PD-1 on B cells and their subtypes was found to be altered in these patients.
CD19 + PD-1 + B cells in these patients had a higher potency to produce anti-dsDNA or IgG.
Nevertheless, this study had some limitations. First, the interaction of T cells and CD19 + PD-1 + B cells was not investigated. Secondly, the molecular mechanisms underlying the dysregulation of PD-1 pathway in CD19 + PD-1 + B cells were not investigated.    Anti-SmD1, anti-Smith D 1 ; Anti-dsDNA, Anti-double-stranded DNA; Anti-U1snRNP, Anti-U1 small