Cross-reactivity of antibodies against interferon beta in multiple sclerosis patients and interference of the JAK-STAT signaling pathway

Figures

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  Figure 1. Interference of the presence of NAbs positive autologous serum in STAT1 activation and cross-reactivity. Expression of pSTAT1 quantified by phosphoflow in monocytes, CD4+ and CD8+ T lymphocytes from MS patients after stimulation with 1000 IU/mL IFNβ (Avonex, Betaferon or Rebif), in presence of autologous serum with NAbs generated against IFNβ1a (Rebif) received as treatment, and in presence of B18R inhibitor. The activation of pSTAT1 reached after IFNß stimulation decreased significantly in presence of NAbs positive autologous serum compared to the activation without serum in T cells and monocytes. NAbs generated against Rebif, were able to cross-react and to decrease significantly pSTAT1 expression achieved with the other two IFNβ molecules in the three subpopulation analyzed. Data are shown as MFI and percentage of cells expressing pSTAT1. Significant p values are shown with asterisk, *p ≤ 0.05; **p ≤ 0.01.

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  Figure 2. STAT1 phosphorylation measures according to the NAbs titers in PBMC from MS patients. Expression of pSTAT1 determined by phosphoflow in monocytes, CD4+ and CD8+ T lymphocytes from MS patients after stimulation with 1000 IU/mL IFNβ1a (Avonex) in presence of several dilutions of a NAbs positive heterologous serum. As controls, non stimulated cells, IFNβ stimulated cells with B18R inhibitor and IFNβ stimulated cells with a NAbs negative heterologous serum, were included. The activation of pSTAT1 achieved after IFNβ stimulation decreased significantly in presence of the different titers of NAbs compared to the activation without serum in T cells and monocytes. In presence of NAbs negative serum no decrease were detected. Data are shown as percentage of cells expressing pSTAT1. Significant p values are shown with asterisk: *p ≤ 0.05. [NAb+]: positive neutralizing antibodies, [NAb−]: negative neutralizing antibodies.

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  Figure 3. Lineal regression analysis between the percentage of pSTAT1 versus the logarithm of the NAbs titers in CD4+ and CD8+ T cells and in monocytes of each independent patient. R2: Coefficient of Determination (R Squared) p: p-value or probability value of the statistical model. N.S: non significant.

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  Figure 4. Gating strategy to determine pSTAT1 expression on different PBMC subsets. Based on the combination of side (SSC) and forward (FSC) light scatter properties of the acquired events, lymphocyte and monocyte gates were selected (a). T lymphocytes were identified by gating on CD3+ cells (b), then, they were transferred to a new dot plot and were analyzed by a specific antibody against CD8. T CD8+ lymphocytes were identified as CD3+ and CD8+ cells and T CD4+ lymphocytes as CD3+ and CD8- cells (c). In a new dot, monocytes were identified as CD14+ and CD3- cells (d). Overlay histograms from a representative patient that depict CD4+, CD8+ T lymphocytes and monocytes expressing pSTAT1 in the different cells stimulation conditions: unstimulated cells (black line), IFNβ stimulated cells (red line), IFNβ stimulated cells in presence of B18R inhibitor (green line), IFNβ stimulated cells in presence of NAbs positive serum (blue line), IFNβ stimulated cells in presence of NAbs negative serum (blue dashed line) and isotype control (shaded histograms) (e). PBMC: peripheral blood mononuclear cells, FSC: forward scatter, SSC: side scatter, PE-Cy7: phycoerythrin-cyanine, PerCP: peridin chlorophyll…