CD19 as a therapeutic target in a spontaneous autoimmune polyneuropathy

Abstract

Spontaneous autoimmune polyneuropathy (SAP) in B7-2 knock-out non-obese diabetic (NOD) mice is mediated by myelin protein zero (P0)-reactive T helper type 1 (Th1) cells. In this study, we investigated the role of B cells in SAP, focusing on CD19 as a potential therapeutic target. We found that P0-specific plasmablasts and B cells were increased in spleens of SAP mice compared to wild-type NOD mice. Depletion of B cells and plasmablasts with anti-CD19 monoclonal antibody (mAb) led to attenuation of disease severity when administered at 5 months of age. This was accompanied by decreased serum immunoglobulin (Ig)G and IgM levels, depletion of P0-specific plasmablasts and B cells, down-regulation/internalization of surface CD19 and increased frequency of CD4(+) regulatory T cells in spleens. We conclude that B cells are crucial to the pathogenesis of SAP, and that CD19 is a promising B cell target for the development of disease-modifying agents in autoimmune neuropathies.

Keywords: B cells; CIDP; Guillain-Barré syndrome; autoimmunity; co-stimulatory molecules.

Fig. 1

Increased frequency of protein zero (P0)-specific plasmablasts (PB) and B cells in spleens of spontaneous autoimmune polyneuropathy (SAP) mice. (a) Antigen-specific PB. Splenocytes with forward- and side-scatter properties of lymphocytes were analysed for CD138 and CD44 to identify PB, followed by selection based on B220 staining and binding of labelled antigens. Gating on antigen-specific PB was set only on strongly positive cells, whose binding was eliminated by pre-blocking for 1 h with unlabelled P0-extracellular domains (ECD). Mean ± standard error of the mean (s.e.m.) percentages of P0-specific PB are indicated in the scatterplots [n = 6 mice each, P < 0·0005 for SAP versus wild-type non-obese diabetic mice (WT NOD)]. (b) Antigen-specific splenic B cells [immunoglobulin (Ig)M⁺B220⁺, IgG⁺B220⁺]. Splenic lymphocytes were initially gated based on IgM and B220 or IgG and B220 staining, followed by selection based on the binding of labelled antigens. Scatterplots shown are from SAP mice. The mean (± s.e.m.) percentages of B cells reactive to P0-ECD in spleens of SAP mice are indicated in the histograms (n = 3–5 each, P < 0·00002 for both P0-ECD-reactive IgM⁺ B cells, and P0-ECD-reactive IgG⁺ B cells comparing SAP versus WT NOD). Acetylcholine receptor (AChR) and ovalbumin (OVA) were used as irrelevant antigens in (a) and (b). (c) Western blot analysis of purified His-tagged P0-ECD protein detected by horseradish peroxidase (HRP)-conjugated goat anti-6-His antibody (1:10 000).

Fig. 2

Anti-CD19 monoclonal antibody (mAb)-mediated effector function and B cell depletion in peripheral blood. (a) Antibody-dependent cell-mediated cytotoxicity (ADCC). The lactate dehydrogenase (LDH) release assay was performed using A20 cells (murine B cell lymphoma) as target cells, and J774·2 cell lines (murine monocytic cell line) or purified mouse natural killer (NK) cells as effector cells (n = 4). Effector cell/target cell ratio: 2·5:1. (b) Complement-dependent cytotoxicity (CDC). A20 cells were incubated with anti-CD19 mAb or isotype control at various concentrations in the presence of mouse serum (source of complement) (n = 3). (c) Acute depletion of peripheral blood B cells and plasmablasts (PB) by anti-CD19 mAb (250 μg) administered intravenously (i.v.) thrice every other day. Experiments were performed on day 8. Total lymphocytes were used for gating based on immunoglobulin (Ig)M and B220 for B cells, and CD138 and B220 for PB. Mean ± standard error of the mean (s.e.m.) percentages are indicated in the scatterplots. Comparing control mAb versus anti-CD19, P < 0·00001 for B cells, P < 0·009 for PB (n = 4–6). (d) Time–course of B cell depletion in the blood using the pulse treatment paradigm. Arrows: i.v. injections with control mAb or anti-CD19 mAb (250 μg) thrice every other week (n = 4). Blood samples were obtained by serial retro-orbital bleeding. The first treatment was given the day after the baseline sample was collected. (e) Reduction in serum IgG and IgM levels by pulse treatment with anti-CD19 mAb. Data from 8 weeks and 12 weeks post-treatment initiation were pooled together and shown as mean ± s.e.m. [**P < 0·0014 for IgM and ***P < 0·00001 for IgG (n = 9 each)].

Fig. 3

Attenuation of disease severity by B cell depletion with anti-CD19 mAb using the pulse treatment paradigm. (a) Mean clinical score [± standard deviation (s.d.)] over the time–course of SAP starting at 5 months (designated as week 0 here). Arrows: intravenous (i.v.) injection with control (Ctrl) mAb or anti-CD19 mAb (250 μg). For data points up to 8 weeks, n = 21 for Ctrl mAb and n = 18 for anti-CD19 mAb, then n = 10 each for data points beyond 8 weeks post-treatment initiation. *P < 0·000001 between two groups at 8 weeks and beyond. (b) Individual clinical scores at 8 and 12 weeks post-initiation of treatment. Horizontal bars represent the median clinical score (**P < 0·0006 for 12 weeks and ***P < 0·00001 for 8 weeks). (c) Grip strength measurements at the end of study period. Data are shown as ± standard error of the mean (s.e.m.) (n = 8–10 each; **P < 0·00001 for both hindlimb and forelimb). (d) Representative examples of sciatic motor response, demonstrating prolonged distal latency (DL), temporal dispersion, slowed conduction velocity (CV) and decreased distal amplitude (dAMP) in recordings from the Ctrl mAb group, but not in those from the anti-CD19 mAb group. (e) Bar graphs summarizing the electrophysiological data, shown as mean ± s.e.m. (n = 8–10 each). *P < 0·0004 for dAMP, **P < 0·00004 for both DL and CV.

Fig. 4

Changes in B cells, plasma cells (PC) and plasmablasts (PB) induced by anti-CD19 mAb using pulse treatment paradigm. (a) The extent of depletion of B cells (IgM⁺B220⁺), PB (B220⁺CD138^hi), PC (B220^loCD138^hi) in the blood, bone marrow (BM) and spleen of spontaneous autoimmune polyneuropathy (SAP) mice studied at 12 weeks. Gating strategy as described in Fig. 1b and Fig 2c. Mean ± standard error of the mean (s.e.m.) percentages are indicated in the scatterplots (n = 4–5 each; P < 0·01 for BM B cells, P < 0·002 for splenic B cells, and P < 0·00002 for blood B cells, P < 0·03 for PB in BM, P < 0·004 for PC in BM and PB in blood, P < 0·0001 for PB and PC in spleen). (b) Data summary from animals studied at 8 weeks, shown as mean ± s.e.m. (n = 5–7). **P < 0·002 for B cells in BM and spleen, ***P < 0·00001 for PB and PC in BM and spleen. (c) Down-regulation/internalization of surface CD19 in splenic B cells at 12 weeks after pulse treatment with anti-CD19 mAb. Lymphocytes were first gated based on B220 and immunoglobulin (Ig)M staining, followed by histograms of CD19 fluorescence. Mean fluorescence intensities (MFI) ± s.e.m. for surface CD19 was 3330·2 ± 50·0 for control mAb treatment, and 1105 ± 30·1 for anti-CD19 treatment (n = 4 each, P < 0·00001). Mean fluorescence intensity (MFI) ± s.e.m. for intracellular CD19 was 3250·5 ± 99·2 for control mAb treatment, and 3116·0 ± 66·3 for anti-CD19 treatment (n = 4 each, P > 0·05).

Fig. 5

Effect of anti-CD19 mAb (pulse treatment) on B cell and T cell responses to myelin protein zero (P0). (a) Scatterplots showing the depletion of P0-specific plasmablasts (PB) by anti-CD19 mAb (n = 3, P < 0·0008). Gating strategy as described in Fig. 1a. (b) Data summary on the depletion of P0-reactive B cells by anti-CD19 mAb (n = 3). *P < 0·004 for P0-specific IgM⁺ B cells; **P < 0·0004 for P0-specific IgG⁺ B cells. (c) Treatment with anti-CD19 mAb did not alter splenocyte proliferation in response to P0 (180–199) and P0-extracellular domains (ECD (ECD) (n = 5 each, P > 0·05). Ovalbumin (OVA) was used as an irrelevant antigen. Data in (a–c) shown as mean ± standard error of the mean (s.e.m.) are from 8 weeks post-treatment initiation.

Fig. 6

Effect of B cell depletion with anti-CD19 mAb on splenic CD4⁺ regulatory T cells (Tregs), CD1d⁺CD5⁺ B cell subset and B10 cells. (a) Increased frequency of splenic Tregs in B cell-depleted spontaneous autoimmune polyneuropathy (SAP) mice. Tregs were identified as CD4⁺CD25⁺forkhead box protein 3 (FoxP3⁺) cells. Data summary includes two acute depletion and four pulse treatment experiments. **P < 0·0003 for %Tregs. (b) Effect of anti-CD19 mAb treatment on B220⁺CD1d⁺CD5⁺ cells and IL-10⁺ B cells (B10 cells) in spleens of SAP mice. Summary for B220⁺CD1d⁺CD5⁺ cells includes three acute depletion and three pulse treatments (12 weeks, **P < 0·0002). For B10 cells, splenocytes were stimulated for 4 h ex vivo with lipopolysaccharide (LPS) (10 μg/ml) and leucocyte activation cocktail containing phorbol myristate acetate (PMA), ionomycin, brefeldin and BD Golgiplug prior to staining with anti-B220 and anti-interleukin (IL)-10 antibodies. *P < 0·003, n = 3 each. Bar graphs in (a) and (b) are shown as mean ± standard error of the mean (s.e.m.).