Fig. 5

RV combined with a PI induces immune activation. (A) Violin plots representing multiparametric flow cytometry studies on PB from MM relapsing patients enrolled in RV + CFZ Phase 1b clinical trial, showing overall expansion of total monocytes absolute count (∗∗p ≤ 0.01) (A); (B) Line graphs representing longitudinal multiparametric flow cytometry studies on PB from MM relapsing patients enrolled in RV + CFZ Phase 1b clinical trial, showing overall expansion of total monocytes absolute count. Wilcoxon signed rank p-values: C1D9 > C1D1 p-value = 0.054; (C-D) Violin plots representing higher frequency of CD14++  CD16- Classical Monocytes (C) and increase in CD69 activation marker in the total monocytes (p = 0.047) (D) on treatment up to the C1D9 compared to baseline C1D1. Statistical analysis was performed following Wilcoxon signed rank p-values, ∗∗p ≤ 0.01, ∗p ≤ 0.05; (E) Violin plots representing a slight increase in CD69 activation marker in the PB monocyte compartment of n = 4 different patients who were not exposed to CFZ prior the treatment compared to n = 4 different patients who instead were CFZ exposed before starting the therapy; (F-G) Violin plots highlighting the increased CD8 expression (F) and the decreased CD4 expression (G) up to C1D9. Data are expressed as change from baseline %, Wilcoxon signed rank p-values: ∗p ≤ 0.05; (H) Violin plot highlighting a decrease in CD4/CD8 ratio from baseline up to C1D9. Data are expressed as change from baseline %, Wilcoxon signed rank p-values: ∗p ≤ 0.05; (I) In-depth immune profiling of a longitudinal CFZ-resistant patient (RRMM-1) enrolled in RV + CFZ Phase 1b was performed with the Maxpar Direct Immune Profiling System using a dry 30-marker antibody panel. Hierarchical clustering and statistical mapping performed algorithmically via the Cytobank© platform. vi-SNE analysis (iterations = 1000, perplexity = 30) displayed in 2D plots using the resultant t-SNE 1 and t-SNE 2 dimensions. High-fidelity FlowSOM (“self-organizing map”) (metacluster = 10 and cluster = 100) based on vi-SNE 2D plots showing 22 immune different immune-compartments; (J-K-L) t-SNE heatmap of 3 longitudinal CFZ-resistant patient enrolled in RV + CFZ Phase 1b highlighting expression of selected monocyte population after treatment C1D9 for RRMM-1 (J), C1D16 for RRMM-2 (K), C1D1 for RRMM-3 after 4 h of treatment (L); (M-N-O) heatmaps showing absolute count of the different monocyte compartments of 3 longitudinal CFZ-resistant patient enrolled in RV + CFZ Phase 1b; (P-Q-R) Heatmaps showing overall distribution of naïve and memory Tregs during the course of the therapy