Abnormal distribution of peripheral B1, Tr, USwM and SwM cell subsets in DCM patients
To investigate the difference of total B cells among the three groups, we first analyzed the percentage of CD19+B and CD20+B cells. No significant difference was observed in the percentage of CD19+B cells (4.65 ± 4.00% for DCM group vs. 6.04 ± 3.97% for HF group vs. 5.67 ± 3.78% for HC group, respectively, P = 0.46) (S1 Fig. A, B) and CD20+B cells (4.72 ± 3.42% vs. 6.61 ± 4.72% vs. 5.50 ± 2.76%, P = 0.24) among the three groups (S1 Fig. C, D).
Then we analyzed the distribution of B cell subpopulations in the three groups. Compared with HF and HC groups, the percentage of B1 cells in DCM group (2.47 ± 1.37% vs. 3.62 ± 2.05% vs. 3.62 ± 1.38%, P = 0.02) was significantly lower (Fig. 2A, 2B), whereas the percentage of Tr cells (6.69 ± 4.77% vs. 3.44 ± 2.69% vs. 3.94 ± 2.78%, P = 0.01) was significantly higher (Fig. 2C, 2E). However, no significant difference was founded in the percentage of B1 and Tr cells between HF and HC groups. The percentage of USwM cells (9.68 ± 4.05% vs. 7.61 ± 2.79% vs. 14.78 ± 6.15%, P < 0.01) (Fig. 3A, 3B) and SwM cells (8.67 ± 3.53% vs. 7.65 ± 3.42% vs. 26.27 ± 9.88%, P < 0.01) (Fig. 3D, 3E) were significantly lower in DCM and HF groups than in HC group. Notably, the percentage of SwM and USwM cells in DCM and HF groups were similar. In addition, there was no significant difference in the percentage of naïve B cells (4.21 ± 2.02% vs. 4.92 ± 2.90% vs. 5.16 ± 2.20%, P = 0.35) (Fig. 2E), mature B cells (50.51 ± 14.10% vs. 46.25 ± 15.78% vs. 41.76 ± 12.31%, P = 0.12) (Fig. 2F), DN cells (7.04 ± 3.71% vs. 8.43 ± 4.00% vs. 6.93 ± 3.19%, P = 0.36) (Fig. 3C), PB (1.07 ± 0.96% vs. 0.89 ± 0.83% vs. 1.07 ± 0.88%, P = 0.35) (Fig. 3F), CD19+CD5+ B cells (8.53 ± 4.29% vs. 8.03 ± 5.68% vs. 11.73 ± 6.14%, P = 0.06) (S2 Fig. A, B) and Breg (5.91 ± 5.65% vs. 3.71 ± 3.41% vs. 4.69 ± 2.99%, P = 0.25) (S2 Fig. C, D) among the three groups.
Finally, we investigated whether there was an association between the percentage changes of B1 cells and Tr cells. Correlation analysis found that no significant association was observed between the two kinds of cells in DCM patients (R=-0.127, P = 0.53) (Fig. 2G).
Plasma levels of IgM, IgG and IgG3 were not affected by abnormal distribution of B cell subsets in DCM patients
To determine the effect of the abnormal distribution of B cell subpopulations on plasma immunoglobulin in DCM patients, ELISA was used to analyze the levels of plasma IgM, IgG and IgG3. However, no significant difference of the total plasma IgM (1.67 ± 0.71 vs. 1.75 ± 0.66 vs. 1.75 ± 0.72 mg/ml, P = 0.74), IgG (9.53 ± 4.35 vs. 10.88 ± 3.34 vs. 10.08 ± 5.34 mg/ml, P = 0.59) and IgG3 (2.97 ± 1.57 vs. 2.95 ± 1.31 vs. 2.63 ± 1.73 mg/ml, P = 0.72) levels was observed among the three groups (Fig. 4A).
Then, the correlation between the percentage of B1 cells and Tr cells and plasma immunoglobulin in patients with DCM was further evaluated. Correlation analysis showed that there was also no significant correlation between B1 cells or Tr cells and plasma levels of IgM (R=-0.124, P = 0.54, for B1 cells; R = 0.226, P = 0.26, for Tr cells, respectively), IgG (R=-0.106, P = 0.60; R=-0.121, P = 0.55) and IgG3 (R = 0.011, P = 0.96; R=-0.332, P = 0.07) in DCM patients (Fig. 4B-G).
Plasma levels of IL-10 and TNF-α were not affected by abnormal distribution of B cell subsets in DCM patients
To estimate the effect of disproportion of B cell subsets on plasma cytokines closely related to DCM, the plasma levels of IL-10 and TNF- α were assayed by ELISA. The similar plasma levels of IL-10 (11.6 ± 5.03 vs. 12.47 ± 6.34 vs. 9.69 ± 1.54 pg/ml, P = 0.14) and TNF-α (9.70 ± 7.84 vs. 10.30 ± 8.39 vs. 6.37 ± 2.29 pg/ml, P = 0.14) were found among the three groups (Fig. 5A).
Next, correlation analysis was used to further explore the effect of B1 and Tr cell percentage changes on plasma IL-10 and TNF-α levels in DCM patients. The results indicated that neither B1 cells nor Tr cells were significantly correlated with plasma levels of IL-10 (R=-0.009, P = 0.97; R = 0.242, P = 0.22) and TNF-α (R=-0.031, P = 0.88; R=-0.410, P = 0.06) in patients with DCM (Fig. 5B-E).
B1 cells were significantly decreased in β1-AR antibody-positive patients with DCM
To evaluate the effect of β1-AR autoantibodies on the percentage of B1 and Tr cells and clinical parameters in patients with DCM, β1-AR autoantibodies of all subjects were detected by ELISA. The positive rates of β1-AR autoantibodies in DCM, HF and HC groups were 48.1%, 27.8% and 4.8%, respectively (P < 0.01). Further analysis found that the percentage of B1 cells in patients with β1-AR autoantibody positive DCM was significantly lower than those with β1-AR autoantibody negative (1.84 ± 0.78% vs. 3.05 ± 1.56%, P = 0.02) (Fig. 2B). However, no similar results were found in Tr cells (6.10 ± 6.09% vs. 7.10 ± 3.78%, P = 0.61) (Fig. 2D).
Interestingly, we also found that plasma NT-proBNP (14932.09 ± 1655.47 vs. 5024.36 ± 863.16 pg/ml, P = 0.04) and hs-CRP (7.075 ± 3.187 vs. 3.049 ± 2.939 mg/L, P = 0.01) levels in DCM patients with positive β1-AR autoantibodies were significantly higher than those with negative β1-AR autoantibodies (Fig. 6). However, the plasma levels of IgM (1.80 ± 0.56 vs. 1.57 ± 0.82, P = 0.39), IgG (9.93 ± 3.83 vs. 9.17 ± 4.85, P = 0.64), IgG3(2.94 ± 1.19 vs. 2.99 ± 1.87, P = 0.93), IL-10 (13.04 ± 4.32 vs. 11.02 ± 5.55, P = 0.28) and TNF-α (11.42 ± 7.33 vs. 8.20 ± 8.20, P = 0.27) were similar in the two subgroups (Fig. 4A, Fig. 5A).
B1 cells were negatively correlated with NT-proBNP and positively correlated with LVEF in DCM patients
To investigate the association between the percentage of B1 and Tr cells and the severity and prognosis of DCM, correlation analysis was performed. The percentage of B1 cells in DCM group was negatively correlated with NT-proBNP (R=-0.532, P < 0.01), positively correlated with LVEF (R = 0.457, P = 0.02), and had no correlation with hs-CRP (R = 0.248, P = 0.21) (Fig. 7A-C). However, the correlations were not significant between the percentage of Tr cells and NT-proBNP (R=-0.209, P = 0.23), LVEF (R = 0.029, P = 0.89) and hs-CRP (R=-0.001, P = 0.99) (Fig. 7D-F).