IVIg-induced plasmablasts in patients with Guillain-Barré syndrome

Abstract

Objective: The Guillain-Barré syndrome (GBS) is an acute, immune-mediated disease of peripheral nerves. Plasmablasts and plasma cells play a central role in GBS by producing neurotoxic antibodies. The standard treatment for GBS is high-dose intravenous immunoglobulins (IVIg), however the working mechanism is unknown and the response to treatment is highly variable. We aimed to determine whether IVIg changes the frequency of B-cell subsets in patients with GBS.

Methods: Peripheral blood mononuclear cells were isolated from 67 patients with GBS before and/or 1, 2, 4, and 12 weeks after treatment with high-dose IVIg. B-cell subset frequencies were determined by flow cytometry and related to serum immunoglobulin levels. Immunoglobulin transcripts before and after IVIg treatment were examined by next-generation sequencing. Antiglycolipid antibodies were determined by ELISA.

Results: Patients treated with IVIg demonstrated a strong increase in plasmablasts, which peaked 1 week after treatment. Flow cytometry identified a relative increase in IgG2 plasmablasts posttreatment. Within IGG sequences, dominant clones were identified which were also IGG2 and had different immunoglobulin sequences compared to pretreatment samples. High plasmablast frequencies after treatment correlated with an increase in serum IgG and IgM, suggesting endogenous production. Patients with a high number of plasmablasts started to improve earlier (P = 0.015) and were treated with a higher dose of IVIg.

Interpretation: High-dose IVIg treatment alters the distribution of B-cell subsets in the peripheral blood of GBS patients, suggesting de novo (oligo-)clonal B-cell activation. Very high numbers of plasmablasts after IVIg therapy may be a potential biomarker for fast clinical recovery.

Figure 1

The number and frequency of plasmablasts are increased in the peripheral blood of GBS patients treated with IVIg. B‐cell subsets were gated as indicated (A). Total B cells (CD19⁺ cells; (B), naïve B cells (CD19⁺ IgD⁺ CD27⁻; (C), memory B cells (CD19⁺ IgD⁻ CD27⁺; (D), natural effector B cells (CD19⁺ IgD⁺ CD27⁺; (E) and plasmablasts (CD19⁺/low CD27⁺ CD38⁺; (F) were quantified using flow cytometry. The percent plasmablasts of total B cells is shown in G. An increase in the percent of plasmablasts was also found in an IVIg‐treated patient with a spinal cord infarction (H). A representative IVIg‐treated GBS patients is shown in I. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

IVIg‐induced plasmablasts are enriched for IgG2. Intracellular staining of immunoglobulin classes and subclasses was performed on cryo‐preserved PBMC from GBS patients (n = 20) and healthy controls (n = 20). Plasmablasts (CD19⁺/low CD27⁺ CD38⁺) were gated and expression of all classes and subclasses was observed (A; top panels pretreatment and lower panels posttreatment). No difference was found in IgM (B), IgA (C), and IgG (D) positive plasmablasts before and 1 week after IVIg treatment. Quantification of IgG1 (E), IgG2 (F), and IgG3 (G) subclasses demonstrated a significant shift in IgG subclass usage 1 week after IVIg treatment. Samples with few plasmablasts were excluded. IGG transcripts analyzed by 454 sequencing also indicated a significant shift in unique IGG subclass sequences in GBS patients (n = 3) 1 week posttreatment (H; two‐way ANOVA followed by Bonferroni correction). The fraction of sequences found in IGG2 only, and not shared with other (sub)classes was also increased after treatment (I; data represent means of three patients). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Dominant B‐cell clones are present in GBS patients after treatment with IVIg. RNA was isolated from PBMC before and 1 week after treatment with IVIg and immunoglobulin transcripts were sequenced. Potential clones were identified as the percent of (all) IGG sequences having the same V and D region usage and the same CDR3 length. Within each patient, unique clones are represented by a different color (only clones of >1%). Before treatment, a very dominant clone was identified in patient 1 (A). After treatment, the number of dominant clones (>5%, indicated with asterisks) increased in all patients and dominant clones were more common than in healthy controls (n = 5; B). Analysis of the CDR3 amino acid composition of the most prevalent clone of every patient before and after treatment identified common motifs (data shown as frequency; C). Note that after treatment the most prevalent clones were of the IgG2 subclass. Comparison of the IgG clone frequency from patient 1 before and after treatment revealed only little overlap between clones (D).

Figure 4

The percent of plasmablasts after 1 week of IVIg treatment correlates with the rise in serum IgM and IgG levels. Serum IgM (A–C) and IgG (D–F) levels were determined before and after IVIg treatment and related to the percent of plasmablasts measured after 1 week of IVIg treatment. Absolute IgM/G levels are shown in A and D, the delta IgM/G levels at 1 week in B and E, and the delta IgM/G level at 2 weeks are indicated in C and F. Delta IgM/G levels were calculated compared to pretreatment levels, which were not available for all patients. Patients randomized for a second course of IVIg or placebo were excluded from analysis in C and F.

Figure 5

Plasmablasts after IVIg treatment in relation to clinical outcome in GBS. The absolute number of plasmablasts in relation to the GBS disability score determined 4 weeks after IVIg therapy (A) or the time to improve one grade on the GBS disability scale (C; y‐axis in log2 scale; n = 43). The time to reach independent walking (B) that is a GBS disability score of two, and the time to improvement of one grade on the GBS disability scale (D) was compared in patients with high (≥50,000 cells/mL) and low absolute numbers (<50,000 cells/mL) using logrank testing (n = 42).

Figure 6

Patients with high plasmablast numbers after IVIg treatment have high GM1 IgG titers at onset. Pre‐ and posttreatment sera from GBS patients positive for GM1 IgG antibodies (n = 12) were titrated by ELISA. Data is shown stratified according to low or high plasmablast count assessed 1 week after IVIg treatment (A). A titer of <100 is considered negative. Two‐way ANOVA followed by Bonferroni correction (***P < 0.001). Subclass analysis of anti‐GM1 antibodies (B–E) demonstrated that the majority of patients do not produce anti‐GM1 antibodies of the IgG2 subclass; only one patient is positive for anti‐GM1 IgG2 1 week after treatment (C). The dotted line indicated the cutoff value (ΔOD = 0.2).