Eleven patients (9 male / 2 female, mean age 62 ± 17 years) with CIDP participated in this study. Inclusion criteria were confirmed or probable CIDP (according to the EFNS/PNS criteria (Van Den Bergh et al. 2010)) on recurrent IVIg treatment (every four weeks), the absence of autoantibodies against paranodal junction proteins, vaccination with a COVID-19 mRNA vaccine (Pfizer-BioNTech BNT162b2), and written informed consent for study participation. Exclusion criteria were acute infections or intake of immunosuppressants or systemic corticosteroids within the previous three months. Mean baseline INCAT disability score was 2 ± 2, mean baseline Rasch-built overall disability scale (R-ODS) score was 73 ± 19.

Blood serum samples and peripheral blood mononuclear cell (PBMC) samples were collected at four timepoints: Before vaccination (mean 11 ± 4 weeks before the first vaccine dose), samples were collected before (1st, on the same day) and one week after IVIg infusion (2nd). The mean interval between the first and the second dose of COVID-19 vaccine was 4 ± 1 weeks. Sample collection was repeated two weeks after the second COVID-19 vaccine dose immediately before (3rd) and one week after IVIg infusion (4th).

Blood samples were collected by venipuncture using Sarstedt S-monovettes® (Sarstedt, Nümbrecht, Germany) for serum collection and the BD Vacutainer CPT™ system (BD Biosciences, San Diego, CA, USA) for PBMC isolation.

Serum samples were obtained by centrifugation of whole blood monovettes at 3000 rotations per minute (rpm) for 10 min and then stored at -20 °C for further use.

PBMCs were isolated from the BD Vacutainer CPT™ system according to the manufacturers ‘ instructions before being stored at -80 °C (15 million PBMCs in 100 µl dimethylsulfoxide [DMSO] and 900 µl fetal bovine serum [FBS]).

The following antibodies were used for the analysis of peripheral B and T lymphocytes, monocytes, and myeloid dendritic cells by flow cytometry: CD3 (OKT3)-BV650, CD11c (3.9)-BV510, CD14 (63D3)-PE, CD16 (3G8)-APC/Cy7, CD19 (HIB19)-PE/Cy7, CD27 (O323)-BV785, CD32b/c (S18005H)-APC, CD56 (5.1H11)-PE Dazzle 594, all from Biolegend, San Diego, CA, USA, and CD32a (#2)-FITC (SinoBiological, Beijing, China).

Raw flow cytometry data were acquired on a BD LSR Fortessa™ cytometer (BD Biosciences) and data were analyzed using FlowJo v.10.8.0 software (BD Biosciences).

All flow cytometry experiments included isotype controls and fluorescence-minus-one controls. Doublet cells were excluded from analyses based on forward scatter-A and forward scatter-H. For each experiment, the lymphocyte and the monocyte population were gated using forward and side scatter.

Monocytes were characterized by excluding CD3 and CD19 positive cells and the differential expression of CD14 and CD16, and defined as classical (CD14+CD16−) and non-classical (CD14dimCD16+) monocytes. Thereafter, the expression of pro-inflammatory CD32a or anti-inflammatory CD32b on monocytes was assessed. The whole lymphocyte population was differentiated into T cells (CD3+CD32a+ and CD3+CD32b+), naïve B cells (CD19+CD27−), either expressing CD32a (CD19+CD27−CD32a+) or CD32b (CD19+CD27−CD32b+), or memory B cells expressing the same antigens (CD19+CD27+CD32a+, CD19+CD27+CD32b+). As CD32c is uniquely expressed on NK cells (Veri et al. 2007), only CD32b expression was considered on the cell populations analyzed in this study.

Myeloid dendritic cells (mDCs) were defined as CD11c+ live cells that were negative for CD3, CD14, CD19, and CD56 (Suppl. Figure 1).

Serum samples were analyzed for IL-6, IL-10, IL-33, MIP-1α, and TGF-β concentrations using sandwich enzyme-linked immunosorbent assay (ELISA) kits (ImmunoTools, Friesoythe, Germany, for IL-6 and IL-10) and Invitrogen human IL-33, MIP-1α, and TGF-β sandwich ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers ‘ instructions. Plates were read at 450 nm and optical densities were analyzed using the BMG Labtech MARS® software (BMG Labtech, Ortenberg, Germany). Serum cytokine concentrations were calculated by interpolating the individual sample’s optical density with a standard concentration curve.

Statistical analysis was performed using GraphPad PRISM 9.0 software (Graphpad, San Diego, CA, USA). Normality was tested using D’Agostino and Pearson omnibus normality test, before either multiple paired t-tests or Wilcoxon tests were performed. A p-value < 0.05 was considered statistically significant.