Rituximab specifically depletes short-lived autoreactive plasma cells in a mouse model of inflammatory arthritis

Abstract

There is increasing appreciation of the important role of B cells in many autoimmune diseases and consequently, increasing interest in treating these disorders through B cell-depletion therapy with rituximab, an anti-CD20 monoclonal antibody. Yet, precisely how this and related drugs exert their therapeutic effects remains controversial. In particular, it is unclear how, in a number of contexts, rituximab can greatly reduce the titer of serum autoantibodies without substantially altering the overall antibody titer. We have studied the action of this drug in the K/BxN mouse model of inflammatory arthritis after first crossing in a human CD20 transgene. Rituximab treatment of these mice led to a decrease in the titer of serum antibodies targeting glucose-6-phosphate isomerase, the relevant autoantigen, but not in the total antibody titer. Glucose-6-phosphate isomerase-specific plasma cells did not reside primarily in the bone marrow as expected but rather in the spleen and lymph nodes, where they had short lives, expressed CD20, and were rapidly depleted by rituximab. These data support a model whereby autoreactive plasma cells (at least certain specificities thereof) are intrinsically different from protective antimicrobial plasma cells in their differentiation, migration, and survival properties. Rituximab targets the former and spares the latter.

Fig. 1.

Ab titers after rituximab treatment. hCD20/K/g7 and K/g7 arthritic mice beginning at 5–8 weeks of age were injected with 1 mg rituximab weekly for up to 18 weeks; some mice were killed early for other experiments). Tail blood samples were collected each week to assay for serum-Ab titers by ELISA. The titer was determined as described in Materials and Methods. (Upper) Anti-GPI IgG titers. (Lower) Total IgG titers.

Fig. 2.

(A) Anti-GPI IgG ASCs in different organs. Cells prepared from the indicated organs of 8-week-old K/BxN arthritic mice and BxN negative-control littermates were assayed by ELISPOT. The mean and SD are shown (n = 3). Cells were from peritoneal cavity exudates (PCE), bone marrow (BM), peripheral blood (PB), spleen (SP), and lymph nodes (LN). (B) Depletion of B cells and ASCs in SP and LNs of hCD20/K/g7 mice after rituximab treatment. Results from five independent experiments with the indicated lengths of drug administration to hCD20-positive and -negative littermates are shown. Each dot represents an individual mouse. The significance of the differences between data on hCD20/K/g7 (filled circles) and K/g7 (open circles) mice was calculated by one-way ANOVA using Stata.

Fig. 3.

Detection of GPI-specific plasma cells by flow cytometry. Cells prepared from SP or LNs of K/BxN or BxN mice at 8 weeks of age were first stained with anti-CD138 mAb and then stained intracellularly with Alexa Fluor 647-labeled GPI. Three gates and the percentages of cells within them are shown after gating on live cells. Representative of five independent experiments.

Fig. 4.

Phenotyping of GPI-specific plasma cells. Each population of cells from 7-week-old K/BxN or BxN mice was defined as in Fig. 3 [GPI intracellular staining (G)]. The number in each histogram is the mean fluorescence intensity (MFI) of all cells or gated cells when the gate is indicated by a bar. Representative of three independent experiments.

Fig. 5.

Depletion of GPI-specific plasma cells after rituximab treatment. (A) Representative flow cytometrical detection of GPI-specific plasma cells. K/g7 and hCD20/K/g7 mice were treated for 3.5 weeks beginning at 7 weeks of age. Cells prepared from SP and LNs are shown. (B) Absolute numbers of the three populations of cells in SP and LNs as defined in A after treatment [GPI intracellular staining (G)]. Lymphocytes were excluded from the G^-CD138⁺ population by forward-scatter and side-scatter. Plotted are results from four independent experiments with 4- to 7-week-old mice treated with rituximab for 1–10 weeks. Each dot represents one individual mouse. Unpaired t test was performed on each population.

Fig. 6.

Expression of human and murine CD20 on plasma cells. Each population of cells from 7-week-old hCD20/K/g7 vs. hCD20/g7 mice or K/BxN vs. BxN mice was defined as in Fig. 3 [GPI intracellular staining (G)]. The number in each histogram represents the MFI of all cells or gated cells when the gate is indicated by a bar. For hCD20 expression, K/g7 mice were used as negative control and indicated by the light dotted line in each histogram. Lymphocytes were excluded from the G^-CD138⁺ population by forward-scatter and side-scatter.

Fig. 7.

Half-life of GPI-specific plasma cells. Eight-week-old K/BxN and BxN mice (n = 2–5 for each time point) were fed BrdU (0.8 mg/mL) in the drinking water for the indicated amount of time. The percentage of BrdU⁺ cells (mean + SD) in each population defined as in Fig. 3 is plotted [GPI intracellular staining (G)]. Lymphocytes were excluded from the G^-CD138⁺ population by forward-scatter and side-scatter.