Belimumab promotes negative selection of activated autoreactive B cells in systemic lupus erythematosus patients

Abstract

Belimumab has therapeutic benefit in active systemic lupus erythematosus (SLE), especially in patients with high-titer anti-dsDNA antibodies. We asked whether the profound B cell loss in belimumab-treated SLE patients is accompanied by shifts in the immunoglobulin repertoire. We enrolled 15 patients who had been continuously treated with belimumab for more than 7 years, 17 matched controls, and 5 patients who were studied before and after drug initiation. VH genes of sort-purified mature B cells and plasmablasts were subjected to next-generation sequencing. We found that B cell-activating factor (BAFF) regulates the transitional B cell checkpoint, with conservation of transitional 1 (T1) cells and approximately 90% loss of T3 and naive B cells after chronic belimumab treatment. Class-switched memory B cells, B1 B cells, and plasmablasts were also substantially depleted. Next-generation sequencing revealed no redistribution of VH, DH, or JH family usage and no effect of belimumab on representation of the autoreactive VH4-34 gene or CDR3 composition in unmutated IgM sequences, suggesting a minimal effect on selection of the naive B cell repertoire. Interestingly, a significantly greater loss of VH4-34 was observed among mutated IgM and plasmablast sequences in chronic belimumab-treated subjects than in controls, suggesting that belimumab promotes negative selection of activated autoreactive B cells.

Keywords: Autoimmunity; B cells; Immunology; Tolerance.

Figure 1. Most B cell subsets are depleted after chronic belimumab therapy.

PBMCs from healthy donors (n = 13), lupus controls (n = 17), and chronic belimumab–treated subjects (n = 15) were stained with a cocktail of antibodies (Supplemental Table 1 – Panel 1) and analyzed by flow cytometry. Cells were gated as shown in Supplemental Figure 1. (A and B) Plots display frequency (A) and absolute cell count/ml (B) of CD19⁺ B cells in gated live singlet lymphocytes. (C–F) Plots display frequency (C and E) and absolute cell count/ml (D and F) of major B cell subsets in gated CD19⁺ B cells. Average percentage depletion of each cell subset compared with lupus controls is shown above the plots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Comparisons were performed using Kruskal-Wallis test (A, C, and E) and Mann-Whitney analysis (B, D, and F).

Figure 2. Belimumab regulates survival of B cells as they transition from the T1 stage to the naive phenotype.

Mature B cells were stained with a cocktail of antibodies (Supplemental Table 1 – Panel 2), and gated as shown in Supplemental Figure 1. Cells were subsetted on the basis of CD24 and CD38 expression and the ability to extrude MitoTracker Green (MTG). (A and B) Plots display frequency (A) and absolute cell count/ml (B) of transitional type 1 (T1), T2, T3, naive, activated naive (29), and MTG⁺CD24⁺CD38^– subsets in gated CD19⁺ B cells. The number of subjects in each group was healthy donor (n = 13), lupus control (n = 14), chronic belimumab (n = 14). (C and D) Pseudocolor plots showing changes in B cell subsets of a representative lupus donor before (C) and after (D) belimumab therapy. Samples with fewer than 200 total B cells collected were excluded from some of the subset analyses due to insufficient cells in the subgates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Comparisons were performed using Kruskal-Wallis test (A) and Mann-Whitney analysis (B).

Figure 3. VH repertoire of unmutated IgM sequences from mature B cells.

(A) Frequency of VH usage of the functional unmutated IgM repertoire from SLE patients treated with chronic belimumab (CB, blue) and lupus controls (LC, green) is shown. Each data point represents an individual subject. Heatmaps show the average frequency of VH-JH paired usage of LC and CB groups (B, left and right, respectively). (C) Clustered heatmap shows the amino acid usage of the CDR3 from LC, CB, reference non-autoreactive, and ANA-reactive groups, and of 5 individuals before (Pre) and 6 months after (Post) belimumab treatment. Distribution of CDR3 physicochemical properties (acidic [D], basic [E]) for LC and CB groups compared with the reference non-autoreactive and ANA-reactive groups. **P < 0.01; ns, not significant (linear mixed model). (F) Distribution of CDR3 amino acid length usage of LC (green) and CB (blue) groups. (G) Principle component analysis (PCA) of Kidera factors. For A, D, and F, the number of subjects in each group was LC (n = 12) and CB (n = 15).

Figure 4. VH repertoire of mutated mature IgM sequences and plasmablasts.

Changes in the VH usage of mutated mature (Mut) versus unmutated mature (Mat) IgM sequences of individual lupus control (LC) (n = 12) and chronic belimumab (CB) patients (n = 14) are shown as ratios in A and B, respectively. Each dot represents one patient. Changes in the VH usage of plasmablast (PB) versus unmutated mature (Mat) of and mutated mature (Mut) of individual CB patients are shown as ratios in C and D, respectively. Only those VH with statistically significant differences performed using a linear mixed model with crossing between cell populations and treatment groups to look for interactions are shown (see Supplemental Table 3).

Figure 5. VH4-34 and CDR3 usage across groups.

Frequency of VH4-34 in each subset for (A) lupus control (LC) (n = 6–8) and (B) chronic belimumab (CB) (n = 13) patients are shown in connected dot plots, with each line representing 1 patient. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Comparisons were performed using Wilcoxon’s rank-sum test. (C) Plot displays the percentage reduction of VH4-34 usage in mutated mature B cells compared with unmutated mature B cells. Comparisons were performed using Mann-Whitney analysis. (D) Clustered heatmap shows the amino acid usage of the CDR3 from unmutated, mutated, and plasmablast (PB) sequences of LC and CB compared with reference non-autoreactive and autoreactive sequences. (E and F) Principle component analysis (PCA) of CDR3 amino acid composition (E) and physicochemical properties (Kidera factors, F) are shown for unmutated, mutated, and PB sequences.