Article Figures & Data
Figures
- Figure 1 Treatment with IVIg reduces NK cell frequency, but not the frequency of T cells and B cells
Flow cytometry analysis of PBMCs from patients with CIDP (n = 14) before treatment initiation and 24 hours after IVIg treatment. (A) Representative gating strategy to define NK cells. Lymphocytes were identified based on the size and granularity of cells. To exclude NKT cells, CD56 and CD3 costaining was performed. Gated on CD56+/CD3− cells, subpopulations of CD16+/CD56dim and CD16−/CD56bright NK cells were determined. (B) Changes in NK cells gated on lymphocytes in individual IVIg-naive patients and 24 hours after IVIg infusion. (C) Proportion of CD56bright and CD56dim NK cell subpopulations within lymphocytes. Depicted is mean ± SEM of the analyzed patients. (D) Changes in CD56bright subpopulation gated on NK cells in individual IVIg-naive patients and 24 hours after IVIg infusion. (E) Changes in frequencies of T cells and B cells gated on lymphocytes in individual IVIg-naive patients and 24 hours after IVIg infusion. (F) Depicted are changes in the ratio of lymphocytes and monocytes within the leukocyte population in individual IVIg-naive patients and 24 hours after IVIg infusion (asterisks indicate significance: ***p < 0.001; nonparametric distribution; Wilcoxon rank test for paired samples). CIDP = chronic inflammatory demyelinating neuropathy; IVIg = IV immunoglobulin; NK = natural killer; PBMC = peripheral blood mononuclear cell.
- Figure 2 Correlation of INCAT with NK cells and their markers
Correlation of the INCAT score with the percentage of NK cells within lymphocytes (A, n = 16) and the expression level of NK-specific genes CD56 and CD94 (B, n = 27). Patients were classified in terms of the INCAT disability score. Depicted is mean ± SD and correlation with Spearman correlation coefficient r, significance level p is as stated. INCAT = Inflammatory Neuropathy Cause and Treatment; NK = natural killer.
- Figure 3 Treatment with IVIg reduces NK cells and NK-specific markers in a reversible manner
Analysis of changes in relative expression levels of the indicated typical NK cell–specific genes in PBMCs from patients with CIDP before treatment initiation and 24 hours after IVIg treatment. (A) Depicted are changes of relative expression of indicated genes in individual patients (n = 17) related to the house keeping gene GAPDH (2−ΔCt, asterisks indicate significance: *p < 0.05, **p < 0.01, ***p < 0.001; nonparametric distribution, Wilcoxon rank test for paired samples). Changes of the indicated genes. (B) Representative expression levels of the indicated genes of a representative patient over time before IVIg treatment initiation and 24 hours after monthly infusion of IVIg. CIDP = chronic inflammatory demyelinating neuropathy; IVIg = IV immunoglobulin; NK = natural killer; PBMC = peripheral blood mononuclear cell.
- Figure 4 In treatment-naive patients, no difference in NK cell marker expression is detectable between responders and nonresponders
Comparison of NK cell markers of PBMCs from patients with CIDP before IVIg treatment that were subsequently classified as responders (n = 11) or nonresponders (n = 6) depending on the treatment efficacy of IVIg. Depicted is mean ± SEM. (A) Changes of frequencies of NK cells, CD56bright and CD56dim NK cell subpopulations, and CD161+ and NKG2A+ populations in patients with CIDP. In addition, mean fluorescence intensity was analyzed of surface protein expression levels of CD161 and CD335. (B) Longitudinal changes in transcriptions levels of the indicated genes after real-time qPCR analysis was performed on mRNA preparations of PBMCs. CIDP = chronic inflammatory demyelinating neuropathy; IVIg = IV immunoglobulin; NK = natural killer; mRNA = messenger RNA; PBMC = peripheral blood mononuclear cell.
- Figure 5 Reduction of NK cell marker expression after 24-hour IVIg treatment is more pronounced in responding patients
(A) Changes of frequencies of relevant NK cell markers in PBMCs of patients with CIDP 24 hours after IVIg infusion related to baseline in responders (n = 11) and nonresponders (n = 6). Frequency of CD56bright NK cells was determined within the total NK cell population and within lymphocytes. Asterisks above bars indicate significant changes after IVIg treatment compared with control (*p < 0.05, **p < 0.01, ***p < 0.001; nonparametric distribution, Wilcoxon rank test for paired samples). Asterisks between bars indicate significant changes between the responder and nonresponder cohort. Depicted is mean ± SEM (*p < 0.05, **p < 0.01; nonparametric distribution, unpaired; Mann-Whitney U test). (B) Changes of the transcription level of relevant NK cell genes in PBMCs of patients with CIDP 24 hours after IVIg infusion related to baseline in responders (n = 11) and nonresponders (n = 6). Asterisks above bars indicate significant changes after IVIg treatment compared with control (*p < 0.05, **p < 0.01; nonparametric distribution, Wilcoxon rank test for paired samples). Asterisks between bars indicate significant changes between the responder and nonresponder cohort. Depicted is mean ± SEM (*p < 0.05, **p < 0.01; nonparametric distribution, unpaired; Mann-Whitney U test). (C) Changes in PBMCs of patients with CIDP after 24 hours of IVIg infusion in representative markers of NK cells on gene expression and surface expression in the individual patients related to baseline (responders n = 8, nonresponders n = 6). CIDP = chronic inflammatory demyelinating neuropathy; IVIg = IV immunoglobulin; NK = natural killer; PBMC = peripheral blood mononuclear cell.
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