Rituximab does not reset defective early B cell tolerance checkpoints

Abstract

Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti-B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti-B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti-B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti-B cell therapy.

Figure 3. Mature naive B cells after rituximab treatment are newly generated clones.

(A) Evaluation of the number of cell divisions undergone in vivo by KREC analysis in new emigrant/transitional (left) and mature naive (right) B cells from a healthy donor and T1D patients before and after treatment with rituximab. (B) Evaluation of the proliferative state in vivo by Ki67 staining of new emigrant/transitional (left) and mature naive (right) B cells from a healthy donor and T1D patients before and after treatment with rituximab. Bold horizontal lines represent averages, and the number of individuals analyzed is indicated. **P < 0.01 and ***P < 0.001, by 2-tailed Student’s t test.

Figure 2. High frequencies of autoreactive B cells in T1D patients after anti–B cell therapy.

(A) Abs cloned from mature naive B cells from a healthy donor (HD27) and 4 T1D patients before and after rituximab treatment were tested by ELISA for HEp-2 cell reactivity. Dotted lines represent the ED38⁺ control, and solid lines show binding for each cloned recombinant Ab. Horizontal lines define the cutoff OD_405nm for positive reactivity. Frequency of HEp-2 cell–reactive clones (solid black area) is summarized in the pie charts, with the total number of clones tested indicated in the centers. Frequencies of HEp-2–reactive (B), polyreactive (C), and antinuclear (D) mature naive B cells were higher in T1D patients compared with frequencies in the healthy donor and remained unchanged after treatment with rituximab. *P < 0.05 and ***P < 0.001, by 2-tailed unpaired Student’s t test for the healthy donor versus T1D patients. HD, healthy donor.

Figure 1. Rituximab treatment results in transient reduced numbers of peripheral B cells.

(A) Populations of lymphocytes from 19 T1D patients 0, 13, 26, and 52 weeks after rituximab treatment were assessed by flow cytometry. CD3, CD3⁺ T cell counts; Tregs, CD3⁺CD4⁺CD25^hiCD62L⁺ cell counts; CD19CD38, CD24^+/–IgD^+/–CD38⁺CD19⁺CD10^+/– cell counts; CD19CD38IgD, CD24^+/–IgD⁺CD38⁺CD19⁺CD10^+/– cell counts; CD19CD27, CD1c^+/–IgD^+/–CD27⁺CD19⁺IgM^+/– cell counts; CD19, CD1c^+/–CD95^+/– CD27^+/–CD19⁺CD20^+/– cell counts; Naive B, CD24⁺IgD⁺CD38^–CD19⁺CD10^– cell counts. Data represent the mean ± SEM. (B) Comparative frequencies of CD19⁺CD10⁺IgM^hiCD27^– new emigrant/transitional B cells (top left), CD19⁺CD10^–IgM⁺CD27^– mature naive B cells (top right), CD19⁺CD10^–CD27⁺ memory B cells (bottom left), and switched memory B cells (bottom right) were determined for T1D patients before and after treatment with rituximab. Bold horizontal lines represent averages, and the number of T1D patients analyzed is indicated. *P < 0.05 and **P < 0.01, by 2-tailed paired Student’s t test. Tx, treatment.