Fig 2: IL-32 expression promotes a more immature plasma cell phenotype(A) Venn-diagram of overlapping significant genes (p <0.01) that were more highly expressed in WT cells compared with KO cells (comparing two INA-6 KO clones [KO1, KO2] with WT mock cells) and upregulated in IL-32 patients (comparing IL-32- expressing patients versus nonexpressing patients). See also Table S5.(B) Gene expression of MME, IRF8, and SORL1 in patients expressing IL-32 (10th percentile) compared with nonexpressing (90th percentile) patients. Significance determined by limma in R.(C) Gene expression of MME, IRF8, and SORL1 in INA-6 IL-32 KO1, KO2, and WT mock cells. Significance determined by limma in R with Benjamini-Hochberg-adjusted p-values. Data presented are mean cpm ± SD of two replicates.(D) Evaluation of gene expression of markers associated with less differentiated stages of B cell maturation in CoMMpass IA13, comparing IL-32 expressing patients (upper 10th percentile) with nonexpressing patients (lower 90th percentile). Significance determined by limma in R. Boxplots show the median and 25th/75th quantiles and smallest/largest value within the 1.5 times interquartile rang.(E) Scatterplot of genes associated with less differentiated stages of B cell maturation in single cells with (N = 142) and without (N = 346) IL32-expression (from single cell transcriptomics). p values were calculated using the FindMarkers function in Seurat by comparing the high and low IL32 groups.(F) Surface expression of CD45 and CD38 in INA-6 KO and WT cells. Data are presented as median fluorescence intensity (MFI) from 3 independent experiments and significance determined by unpaired student's t test. Bare plots show mean ± SEM.(G) Concentration of kappa light chain/cell detected in conditioned media from WT and KO cells as indicated. p values were calculated by the ratio paired t test. ns, not significant; *p =0.05, **p =0.01, ***p =0.001, ****p =0.0001.

Fig 3: ITI-hUC-MSCs secrete C1INH to inhibit CD301+ macrophage polarization(A and B) The proportion of CD301+ macrophages and mean fluorescence intensity (MFI) of CD301 in PMA-treated THP-1 cells stimulated with LPS and IFN-? for 24 h and then treated with conditioned media (CM) from hUC-MSCs (n = 3) and ITI-hUC-MSCs (n = 3) for another 24 h was measured by flow cytometry.(C and D) Heatmap and volcano plot showing DEGs in hUC-MSCs (n = 3) and ITI-hUC-MSCs (n = 3). The red dots represent the upregulated transcripts, and the blue dots represent the downregulated transcripts (FDR (false discovery rate) < 0.05 and fold change >2).(E) Venn analysis of the top 200 genes by FC (fold change), top 200 genes by FDR and top 200 genes by expression abundance.(F) Heatmap showing the 43 genes filtered out through Venn analysis.(G) The mRNA levels of Serping1, Cxcl5, Il32, Il32a, Il32ß, Il32? and Mmp3 in hUC-MSCs (n = 4) and ITI-hUC-MSCs (n = 4) were measured by qRT?PCR.(H) The protein level of C1INH was examined by western blotting in hUC-MSCs (n = 3) and ITI-hUC-MSCs (n = 3).(I) The relative band intensities of C1INH were analyzed by ImageJ.(J and K) The proportion of CD301+ macrophages and MFI of CD301 in PMA-treated THP-1 cells stimulated with LPS and IFN-? for 24 h and treated with 100 ng/mL recombinant C1INH for another 24 h (n = 3) were measured by flow cytometry.(L) The mRNA level of Serping1 in hUC-MSCs (n = 5) transfected with three different small interfering RNA-seq of Serping1 (si-1, si-2, si-3) or negative control (si-NC) for 48 h was examined by qRT?PCR.(M) The protein level of C1INH in hUC-MSCs (n = 3) transfected with si-1, si-2, si-3 or si-NC for 48 h was examined by western blotting.(N) The relative band intensities of C1INH were analyzed by ImageJ.(O and P) The proportion of CD301+ macrophages and MFI of CD301 in PMA-treated THP-1 cells stimulated with LPS and IFN-? for 24 h and treated with conditioned media of ITI-hUC-MSCs transfected with si-NC (n = 3) and si-Serping1 (si-2; n = 3) for another 24 h was measured by flow cytometry. (A-B), (G), (I-L) and (N-P) Error bars indicate the mean ± SD. No significant difference (ns), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: IL-32 expression in primary myeloma cells is associated with inferior survival, cell division, and oxidative phosphorylation(A) Overall survival of IL-32 expressing patients (10th percentile) compared with nonexpressing patients (90th percentile) in the IA13 CoMMpass dataset P = 8.9e-5, using Cox proportional-hazards regression model.(B) IL-32 expression in individual patients at diagnosis and first relapse in RNA-sequenced CD138+ cells from CoMMpass IA13. Significance was determined by Wilcoxon signed-rank test.(C) GO-analysis of the upregulated genes (Benjamini-Hochberg-adjusted p value <0.05; log2 fold change >0 for up-regulated genes) in IL-32-expressing patients (10th percentile) compared with IL-32 nonexpressing patients (90th percentile). Top significantly enriched biological processes upregulated in IL-32 expressing patients are shown. The GO terms are ordered by the Benjamini-hochberg adjusted p values. See also Tables S3 and S4.(D) Correlation between IL32 and a proliferative index gene signature (calculated as the sum of expression values of the gene set as described in Hose et al. (2009).(E) GO-analysis of the downregulated genes (Benjamini-Hochberg-adjusted p value <0.05; log2 fold change <0 for down-regulated genes, respectively) in IL-32-expressing patients (10th percentile) compared with IL-32 nonexpressing patients (90th percentile). Top significantly enriched biological processes downregulated in IL-32 expressing patients are shown. The GO terms are ordered by the Benjamini-Hochberg adjusted p values. See also Tables S3 and S4.