Intravenous Immunoglobulin Elevates Regulatory T Cells in Guillain-Barré Syndrome: A Potential Biomarker of Therapeutic Response

Abstract

Background and aims: Intravenous immunoglobulin (IVIg) is the primary treatment for Guillain-Barré syndrome (GBS), yet its immunological mechanisms underlying variable clinical outcomes remain unclear. This study investigated the immunomodulatory effect of IVIg on regulatory T cells (Tregs), cytokines, and their association with treatment response and clinical outcomes.

Methods: In this prospective case-controlled study, 57 GBS patients and 57 age- and sex-matched healthy controls (HCs) were investigated. CD4⁺CD25⁺FoxP3⁺ Treg percentages, cytokine production (IL-10, TNF-α, IFN-γ, and IL-12), and serum C3 levels were measured using flow cytometry, Luminex assay, and turbidimetric methods, respectively. Treatment response was defined as ≥ 1-point GBS-disability score improvement during evaluation.

Results: GBS patients exhibited lower CD4⁺CD25⁺FoxP3⁺ Tregs frequencies compared to HCs (p = 0.006), which were inversely associated with serum C3 levels (p = 0.003) during the acute phase. At 4 weeks post-onset, patients with normal C3 levels (90-180 mg/dL) exhibited higher Treg frequencies (p = 0.005) compared to acute GBS, whereas patients with persistently elevated C3 levels showed reduced Treg percentage (p = 0.009). Among I VIg-treated patients, Tregs significantly increased at 2 and 4 weeks post-treatment, alongside significantly higher IL-10 and lower TNF-α, IFN-γ, and IL-12 levels at 4 weeks. However, patients with supportive care showed no such changes in Tregs and cytokine levels. Furthermore, Tregs elevated significantly in patients responsive to IVIg at 2 and 4 weeks (p < 0.05), but not in non-responsive or supportive care patients.

Interpretation: IVIg treatment modulates immune dysregulation in GBS by expanding CD4⁺CD25⁺FoxP3⁺ Tregs and altering cytokines and serum C3 levels, which are associated with clinical improvement. These findings indicate Tregs as potential biomarkers for monitoring initial clinical response to IVIg in GBS.

Keywords: Guillain–Barré syndrome; clinical outcomes; intravenous immunoglobulin; regulatory T cells; treatment response.

Figure 1:. Treg frequency differences between patients with GBS and healthy controls (HCs).

(A) Viable lymphocytes were gated using SSC-A, and CD4⁺ T cells were identified. (B) CD4⁺CD25⁺FoxP3⁺ Tregs were distinguished using quadrant gating. (C) The median (IQR) difference in the percentage of CD4⁺CD25⁺FoxP3⁺ Tregs were compared between individuals with acute GBS and HCs, across GBS subtypes (AMAN vs. AIDP), and (D) between patients with or without preceding C. jejuni infection and anti-GM1-antibodies. Data was visualized as scattered-plot with bars indicating medians with horizontal lines and interquartile ranges (IQR). Statistical analysis was performed using the Mann-Whitney test, with significance at p-value < 0.05.

Figure 2:. Immunomodulatory effect of IVIg on Tregs and cytokines response in GBS.

(A) Treg expression throughout the disease course was visualized using ZebraPlot using FlowJo software. (B) Longitudinal changes in CD4⁺CD25⁺FoxP3⁺ Treg percentages following IVIg treatment and supportive care across different timepoints in GBS patients. (C) The median difference of pro- inflammatory (TNF-α, IFN-γ, and IL-12) and anti-inflammatory (IL-10) cytokines were assessed in IVIg-treated patients and (D) supportively-treated patients with GBS between prior treatment and 4 weeks timepoints. Data was visualized as scattered-plot with bars indicating medians with horizontal lines and IQR. The Y-axis was segmented into two parts for C and D panels to accommodate higher range values in the figure. Statistical comparisons of Treg percentages were performed using the Mann-Whitney test, with significance defined as p-value < 0.05.

Figure 3:. Association between Treg responses, complement C3, and the modulatory effects of IVIg in GBS.

(A) Comparison of serum C3 levels between GBS patients and HCs, among GBS patients with or without anti-GM1 antibodies and preceding C. jejuni infection. (B) Spearman’s rank correlation coefficients between CD4⁺CD25⁺FoxP3⁺ Treg frequencies and serum C3 levels in GBS patients. (C) Comparison of CD4⁺CD25⁺FoxP3⁺ Treg levels in GBS patients with normal versus persistently high serum C3 levels at acute GBS (prior-treatment), and at 2- and 4-weeks post-onset. (D) Serum C3 levels at baseline, 2, and 4 weeks after IVIg or supportive care treatment, analyzed using the Kruskal–Wallis test. (E) Correlation matrix showing associations between serum C3 levels, Treg frequencies, and cytokine levels at 4 weeks post-treatment in both IVIg and supportive care groups.

Figure 4:. Association between Treg responses and clinical outcomes following IVIg or supportive care in GBS patients.

(A) Paired t-test analysis showing changes in CD4⁺CD25⁺FoxP3⁺ Treg frequencies between the acute phase (prior-treatment) and follow-up at 2- and 4-weeks in patients who clinically responded to IVIg or supportive care (defined as ≥1-grade improvement in GBS-DS). (B) Paired t-test analysis of Treg changes in treatment non-responsive (patients with no improvement in GBS-DS at 2- or 4-weeks).