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. 2022 Sep 2;3(6):536–553. doi: 10.1158/2643-3230.BCD-21-0217

Figure 6.

Figure 6. Clus2 cells are enriched for inflammatory transcriptional and proteomic programs. A, Panther pathway analysis of genes differentially expressed in Clus2. Clus2 cells showed the most significant association with cytokine receptor signaling and inflammation. B, Analysis of publicly available bulk RNA-seq data sets showed differential regulation of receptors between healthy and CMML CD34+ cells. The shortlisted 51 receptors were evaluated for expression and frequency data using PE-conjugated flow cytometry screen (n = 4 patients, 15 healthy subjects). Based on the data from the screen, 22 cytokine receptors were shortlisted to be included in the 28-color flow cytometry panel. C and D, Comparison of CRD using the Shannon index showed an increase in CRD in myeloid progenitors of patients (CMML) compared with HSCs but not in controls (Ctrls). E, Comparison of CRD between patients and controls showed a higher Shannon index in patient GMPs (P = 0.04), n = 26 patient cases, and five control cases. F, High diversity (HD) patient GMPs showed significantly higher CD120b expression as compared with low diversity (LD) GMPs (P = 0.03), n = 26 patient cases. Data were analyzed using a nonparametric Mann–Whitney test. G, Balloon plot shows MFIs of cytokine receptors in 26 patient cases and five control cases, along with the Shannon index for each patient and control. As highlighted in the red box, the high diversity patient GMPs showed higher CD120b expression. H, Colony-forming assays with BMMNCs derived from high diversity (n = 2) and low diversity cases (n = 2) showed a significantly higher ratio of the total number of colonies per well in the methylcellulose-based medium with recombinant human cytokines/total number of colonies per well in the methylcellulose-based medium without human cytokines in high diversity as compared with low diversity cases (P = 0.02). Data were analyzed using a nonparametric Mann–Whitney test. I, KM survival analysis showed inferior survival in patients with high-diversity GMPs (log-rank P = 0.02), (J) high-diversity triple-positive HSCs (log-rank P = 0.05), and (K) high-diversity double-positive HSCs (log-rank P = 0.02) as compared with patients with the low-diversity counterparts, n = 26 patient cases. Data were analyzed using log-rank (Mantel–Cox) test. P value significance represented by *, < 0.05; **, < 0.01; ***, < 0.001.

Clus2 cells are enriched for inflammatory transcriptional and proteomic programs. A, Panther pathway analysis of genes differentially expressed in Clus2. Clus2 cells showed the most significant association with cytokine receptor signaling and inflammation. B, Analysis of publicly available bulk RNA-seq data sets showed differential regulation of receptors between healthy and CMML CD34+ cells. The shortlisted 51 receptors were evaluated for expression and frequency data using PE-conjugated flow cytometry screen (n = 4 patients, 15 healthy subjects). Based on the data from the screen, 22 cytokine receptors were shortlisted to be included in the 28-color flow cytometry panel. C and D, Comparison of CRD using the Shannon index showed an increase in CRD in myeloid progenitors of patients (CMML) compared with HSCs but not in controls (Ctrls). E, Comparison of CRD between patients and controls showed a higher Shannon index in patient GMPs (P = 0.04), n = 26 patient cases, and five control cases. F, High diversity (HD) patient GMPs showed significantly higher CD120b expression as compared with low diversity (LD) GMPs (P = 0.03), n = 26 patient cases. Data were analyzed using a nonparametric Mann–Whitney test. G, Balloon plot shows MFIs of cytokine receptors in 26 patient cases and five control cases, along with the Shannon index for each patient and control. As highlighted in the red box, the high diversity patient GMPs showed higher CD120b expression. H, Colony-forming assays with BMMNCs derived from high diversity (n = 2) and low diversity cases (n = 2) showed a significantly higher ratio of the total number of colonies per well in the methylcellulose-based medium with recombinant human cytokines/total number of colonies per well in the methylcellulose-based medium without human cytokines in high diversity as compared with low diversity cases (P = 0.02). Data were analyzed using a nonparametric Mann–Whitney test. I, KM survival analysis showed inferior survival in patients with high-diversity GMPs (log-rank P = 0.02), (J) high-diversity triple-positive HSCs (log-rank P = 0.05), and (K) high-diversity double-positive HSCs (log-rank P = 0.02) as compared with patients with the low-diversity counterparts, n = 26 patient cases. Data were analyzed using log-rank (Mantel–Cox) test. P value significance represented by *, < 0.05; **, < 0.01; ***, < 0.001.