CNS Autoimmune Responses in BCMA-Deficient Mice Provide Insight for the Failure of Atacicept in MS

Abstract

Objective: B cells have emerged as a therapeutic target for MS. Anti-CD20 antibodies, which deplete B cells, are effective therapies for MS. However, atacicept (TACI-Fc), which blocks BAFF and APRIL and reduces B cells, unexpectedly exacerbates MS. We tested the hypothesis that B cell maturation antigen (BCMA), a receptor for BAFF and APRIL, plays a role in the paradoxical effects of anti-CD20 antibody and TACI-Fc using experimental autoimmune encephalomyelitis (EAE).

Methods: EAE was induced in wild-type (BCMA^+/+) and BCMA-deficient (BCMA^-/-) mice with an immunization of rodent myelin oligodendrocyte glycoprotein (MOG)_35-55 peptide. Treatment with anti-CD20 antibody, TACI-Fc, and isotype controls was administered by intraperitoneal injections. CNS infiltration was evaluated by histology; immune cell phenotypes were evaluated by flow cytometry; MOG-specific antibodies were determined by ELISA. Mixed bone marrow chimeras and cell culture assays were used to identify the specific subsets of immune cells affected by BCMA deficiency.

Results: First, we found that BCMA^-/- mice had more severe EAE compared with BCMA^+/+ mice and the increased disease was associated with elevated anti-MOG B-cell responses. Second, we found that anti-CD20 therapy attenuated EAE in BCMA^-/- mice but not in BCMA^+/+ mice. Third, TACI-Fc attenuated EAE in BCMA^+/+ mice but not in BCMA^-/- mice. Mixed bone marrow chimeric and cell culture experiments demonstrated that BCMA deficiency elevates inflammatory B-cell responses but inhibits inflammatory responses in macrophages.

Conclusions: BCMA has multifaceted roles during inflammation that affects therapeutic efficacies of anti-CD20 and TACI-Fc in EAE. Our results from BCMA-deficient mice provide insights into the failure of atacicept in MS.

Figure 1. BCMA Deficiency Exacerbated EAE Disease Severity

(A) EAE was induced in BCMA^−/− mice and BCMA^+/+ mice and scored daily for disease severity. Data are pooled from 3 experiments (n = 15–18/group). Nonparametric Mann-Whitney tests were used to determine statistical significance (*p < 0.05 and **p < 0.01). (B) Representative spinal cord sections from BCMA^+/+ and BCMA^−/− mice harvested at EAE day 15. Sections were stained with hematoxylin and eosin (H&E) and Luxol fast blue, and arrows indicate lesions. Dark purple color indicates infiltrating cells, and blue color indicates the myelin. The presence of (C) GM-CSF⁺ CD4 T cells, (D) MHCII⁺GR1⁺ inflammatory macrophages, and (E) GL7⁺IgD⁻ germinal center (GC) B cells and (F) CD138⁺CD38⁺ plasma cells in the spinal cords of BCMA^+/+ and BCMA^−/− mice (EAE day 15) was measured by flow cytometry. Data are pooled from 2 experiments (n = 5–10/group). Error bars represent SEM, and Student t tests were used to determine statistical significance (*p < 0.05 and **p < 0.01). BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis; Ig = immunoglobulin; MHCII = major histocompatibility complex class II.

Figure 2. BCMA Deficiency Increases B Cell Responses Against MOG During EAE

Serum was collected from BCMA^+/+ and BCMA^−/− mice on EAE days 0, 10, and 15, and (A) MOG IgG and (B) total-IgG were measured by ELISA (n = 5 per group). At EAE day 15, absolute numbers of (C) transitional (Trans) B cells, (D) regulatory B cells (Bregs), (E) class-switched memory (CSM) B cells, (F) germinal center (GC) B cells, and (G) plasma cells/plasmablasts (PC/PB) in the lymph node and spleen cells from BCMA^+/+ and BCMA^−/− mice were measured by flow cytometry. (H) The ratio of trans/CSM B cells from lymph nodes and spleens from BCMA^+/+ and BCMA^−/− mice was determined. Data are pooled from 2 experiments (n = 10/group). ^+/+ represents BCMA^+/+, and ^−/− represents BCMA^−/−. Error bars represent SEM, and Student t tests were used to determine statistical significance. p < 0.05 is statistically significant. BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis; Ig = immunoglobulin; MOG = myelin oligodendrocyte glycoprotein.

Figure 3. Anti-CD20 Treatment Ameliorates EAE in BCMA−/− Mice but Not in BCMA+/+ Mice

(A) BCMA^+/+ mice were treated with either isotype control or anti-CD20 on day 5 and day 10 postinduction of EAE and scored daily. Arrows indicate the treatment days. Data are pooled from 2 experiments (n = 10/group). (B) At the peak of the disease, numbers of splenic B cells, spinal cord–infiltrating macrophages, and T helper cells from isotype control and anti-CD20 treated BCMA^+/+ mice were assessed by flow cytometry (n = 4/group). (C) BCMA^−/− mice were treated with either isotype control or anti-CD20 on day 5 and day 10 postinduction of EAE and scored daily. Arrows indicate the treatment days. Data are pooled from 2 experiments (n = 10/group). (D) At the peak of the disease, numbers of B cells in the spleen and numbers of macrophages and T helper cells in the spinal cord from isotype control and anti-CD20–treated BCMA^−/− mice were assessed by flow cytometry (n = 4/group). Error bars represent SEM. Nonparametric Mann-Whitney tests were used for EAE scores (*p < 0.05). Student t tests were used for the flow cytometric data. TACI-Fc treatment ameliorates EAE in BCMA^+/+ mice but not in BCMA^−/− mice. (E) BCMA^+/+ mice were treated with Ig control or TACI-Fc on day 5 and day 10 postinduction of EAE and scored daily. Arrows indicate the treatment days. Data are pooled from 2 experiments (n = 10/group). (F) At the peak of the disease, numbers of splenic B cells, spinal cord–infiltrating macrophages, and T helper cells from control and TACI-Fc treated BCMA^+/+ mice were assessed by flow cytometry (n = 5/group). (G) BCMA^−/− mice were treated with Ig control or TACI-Fc on day 5 and day 10 postinduction of EAE and scored daily. Arrows indicate the treatment days. Data are pooled from 2 experiments. N = 10/group. (H) At the peak of the disease, numbers of B cells in spleen and numbers of macrophages and T helper cells in the spinal cord of control and TACI-Fc–treated BCMA^−/− mice were assessed by flow cytometry (n = 5/group). Error bars represent SEM. Nonparametric Mann-Whitney tests were used for EAE scores (*p < 0.05). Student t tests were used for the flow cytometric data. BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis.

Figure 4. BCMA Directly Affects B Cells and Myeloid Cells During EAE

(A) B cells, CD4 T cells, macrophages, and neutrophils were FACS sorted from BCMA^+/+ mice with EAE, and quantitative real-time PCR was performed for BCMA expression. Data represent 1/ΔCt of BCMA expression on B cells, CD4 T cells, macrophages, and neutrophils (n = 4/group). Analysis of variance was used for statistics (*p < 0.05). (B) Bone marrow chimeric strategy. Lethally irradiated CD45.1 BCMA^+/+ mice were transplanted with equal number of bone marrow cells from CD45.1 BCMA^+/+ and CD45.2 BCMA^−/− (n = 5). Eight weeks after transplantation, EAE was induced. The percentage of B cells, myeloid cells, and T helper cells derived from donor BCMA^+/+ and BCMA^−/− cells in were assessed in (C) the blood before inducing EAE and in the (D) blood and (E) spleens 20 days after EAE induction. (F) The percentage of class-switched memory (CSM) B cells, germinal center (GC) B cells, plasmablasts (PB), transitional (Trans) B cells, regulatory B cells (Bregs) derived from donor BCMA^+/+, and BCMA^−/− cells from spleens during EAE. (G) The percentage of B cells, myeloid cells, and T helper cells derived from donor BCMA^+/+ and BCMA^−/− cells that have infiltrated the spinal cord during EAE. (H) Percentage of inflammatory macrophages and neutrophils derived from donor BCMA^+/+ and BCMA^−/− cells that have infiltrated the spinal cord of EAE mice. Error bars represent SEM, and Student t tests were used to determine statistical significance. BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis.

Figure 5. BAFF and APRIL Signals Through BCMA to Have Anti-inflammatory Functions in B Cells

(A) Four different B cell subsets, CD19⁺IgM^hiIgD^lo (transitional/regulatory B cells), CD19⁺IgD^hi (mature B cells), CD19⁺IgM^loIgD^lo (class-switched memory B cells), CD19^+/-CD138⁺ (plasmablasts/plasma cells), and CD4⁺ T cells were FACS sorted from BCMA^+/+ mice with EAE, and quantitative real-time PCR was performed for BCMA expression. The data represent 1/ΔCt of BCMA expression (n = 4/group). Error bars represent SEM, and analysis of variance was used to determine statistical differences. (B) Trans (transitional/regulatory) B cells, mature B cells, CS-Mem (class-switched memory B cells), and PB/PC (plasmablasts/plasma cells) were FACS sorted from BCMA^+/+ and BCMA^−/− mice and stimulated with anti-CD40 in the presence or absence of BAFF and APRIL. To get enough numbers of smaller B cell subsets, 5 spleens were pooled together. After 72 hours, cell culture supernatants were collected, and IL-6 and IL-10 secretion was measured by ELISA. Error bars represent SEM, and Student t tests were used to determine statistical significance. p < 0.05 was considered to be statistically significant. APRIL = a proliferation-inducing ligand; BAFF = B cell–activating factor; BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis; IL = interleukin.

Figure 6. BAFF and APRIL Signals Through BCMA to Have Proinflammatory Functions in Myeloid Cells

CD11b⁺ myeloid cells were MACS sorted from healthy (A) BCMA^+/+ and (B) BCMA^−/− mice and stimulated with lipopolysaccharides in the presence or absence of BAFF or APRIL (n = 3/group). After 72 hours, supernatants were collected, and IL-6 and IL-10 secretion was measured by ELISA. Error bars represent SEM, and Student t tests were used to determine statistical significance. p < 0.05 was considered to be statistically significant. APRIL = a proliferation-inducing ligand; BAFF = B cell activating factor; BCMA = B cell maturation antigen; EAE = experimental autoimmune encephalomyelitis; IL = interleukin.