Figure 4.
Downregulation of miR-146b-5p expression by CLL cells cocultured with CD40L-TC. (A) Comparison of IL-23R complex expression by cells from a representative CLL case (GE1-AG114) transfected with the indicated miR inhibitors or cocultured with CD40L-TC or mock control cells for 48 hours and analyzed by flow cytometry. (B) Summary of data on IL-23R complex expression (determined by flow cytometry) after a 48-hour exposure of purified CLL cells to miR-146b-5p inhibitor or CD40L-TC as in (A). CLL cells were from cases GE1-AG114, GE1-DM210, GC0015, SV1-SA, SR1-ME1077, MG0482, VF0384, and CM18. P values of the difference between CLL cells treated with miR-146b-5p vs miR CTR inhibitors or exposed to CD40L-TC vs mock cells are indicated (Wilcoxon test). ∗∗P = .0078. (C) Time course experiments to determine cell viability, cell activation, and miR-146b-5p expression following coculture with CD40L-TC. The top panels show CLL cells morphology based on their side scatter-A and forward scatter-A features before (T0) and after CD40L engagement in the representative CLL case GE1-CC190. The gate (live gate) indicates viable cells. Lower panels summarize data of the experiments in 3 different CLL cases (GE1-GA191, GE1-CC190, and SR1-ME1077). Cell viability was measured by flow cytometry by excluding annexin-V/PI-positive cells (left panel). Expression of the CD80 activation marker by purified CLL cells was evaluated by flow cytometry and is indicated as the percentage of positive cells in the viable cell gate (middle panel). The lower right panel shows miRNA expression by RT-qPCR. Data are expressed as ΔCT of miR-146b-5p vs miR-93. Data are plotted as mean ± SD. (D) RT-qPCR analysis of miR-146b 5p (CLL cases PF0024, HG0135, SR0112, CA0058, RD0468, and LG0337) and IL-12Rβ1 mRNA (CLL cases PF0024, HG0135, CA0058, RD0468, MA0151, and AR0090) expression by CLL cells transfected with miR-146b-5p inhibitors and subsequently cultured with CD40L-TC for 48 hours. miR-146b-5p expression was calculated as fold change compared with values observed in CLL cells transfected with miR-CTR inhibitors normalized to RNU44 and U6 small nuclear RNA(left panel). IL-12Rβ1 mRNA expression was calculated as fold change using CLL cells transfected with miR CTR inhibitor cells as calibrator normalized vs POL2RA gene mRNA (right panel). Data are plotted as mean ± SD. (E) IL-12Rβ1 chain expression by CLL cells from cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077 transfected with the indicated miR inhibitors and cultured for 72 hours in the presence of CD40L-TC. Cells were analyzed by flow cytometry, and data are expressed as a percentage of positive cells. (F) IL-23R complex expression by CLL cells of the same cases analyzed in (E). Data are expressed as a percentage of positive cells (mean ± SD). P values of the difference between stimulated CLL cells and control samples are indicated (Wilcoxon test). ∗P < .05. (G) Flow cytometric analysis of IL-21 receptor expression by CLL cells treated as in (E) in a representative case DF0319. (H) Comparison of IL-23R complex or IL-21R expression by CLL cells from 5 cases (HG0135, SR0112, DF0319, RD0468, and PD0164) treated with the indicated miR inhibitors and cultured with CD40L-TC cells. Data are expressed as a percentage of positive cells (mean ± SD).

Downregulation of miR-146b-5p expression by CLL cells cocultured with CD40L-TC. (A) Comparison of IL-23R complex expression by cells from a representative CLL case (GE1-AG114) transfected with the indicated miR inhibitors or cocultured with CD40L-TC or mock control cells for 48 hours and analyzed by flow cytometry. (B) Summary of data on IL-23R complex expression (determined by flow cytometry) after a 48-hour exposure of purified CLL cells to miR-146b-5p inhibitor or CD40L-TC as in (A). CLL cells were from cases GE1-AG114, GE1-DM210, GC0015, SV1-SA, SR1-ME1077, MG0482, VF0384, and CM18. P values of the difference between CLL cells treated with miR-146b-5p vs miR CTR inhibitors or exposed to CD40L-TC vs mock cells are indicated (Wilcoxon test). ∗∗P = .0078. (C) Time course experiments to determine cell viability, cell activation, and miR-146b-5p expression following coculture with CD40L-TC. The top panels show CLL cells morphology based on their side scatter-A and forward scatter-A features before (T0) and after CD40L engagement in the representative CLL case GE1-CC190. The gate (live gate) indicates viable cells. Lower panels summarize data of the experiments in 3 different CLL cases (GE1-GA191, GE1-CC190, and SR1-ME1077). Cell viability was measured by flow cytometry by excluding annexin-V/PI-positive cells (left panel). Expression of the CD80 activation marker by purified CLL cells was evaluated by flow cytometry and is indicated as the percentage of positive cells in the viable cell gate (middle panel). The lower right panel shows miRNA expression by RT-qPCR. Data are expressed as ΔCT of miR-146b-5p vs miR-93. Data are plotted as mean ± SD. (D) RT-qPCR analysis of miR-146b 5p (CLL cases PF0024, HG0135, SR0112, CA0058, RD0468, and LG0337) and IL-12Rβ1 mRNA (CLL cases PF0024, HG0135, CA0058, RD0468, MA0151, and AR0090) expression by CLL cells transfected with miR-146b-5p inhibitors and subsequently cultured with CD40L-TC for 48 hours. miR-146b-5p expression was calculated as fold change compared with values observed in CLL cells transfected with miR-CTR inhibitors normalized to RNU44 and U6 small nuclear RNA(left panel). IL-12Rβ1 mRNA expression was calculated as fold change using CLL cells transfected with miR CTR inhibitor cells as calibrator normalized vs POL2RA gene mRNA (right panel). Data are plotted as mean ± SD. (E) IL-12Rβ1 chain expression by CLL cells from cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077 transfected with the indicated miR inhibitors and cultured for 72 hours in the presence of CD40L-TC. Cells were analyzed by flow cytometry, and data are expressed as a percentage of positive cells. (F) IL-23R complex expression by CLL cells of the same cases analyzed in (E). Data are expressed as a percentage of positive cells (mean ± SD). P values of the difference between stimulated CLL cells and control samples are indicated (Wilcoxon test). ∗P < .05. (G) Flow cytometric analysis of IL-21 receptor expression by CLL cells treated as in (E) in a representative case DF0319. (H) Comparison of IL-23R complex or IL-21R expression by CLL cells from 5 cases (HG0135, SR0112, DF0319, RD0468, and PD0164) treated with the indicated miR inhibitors and cultured with CD40L-TC cells. Data are expressed as a percentage of positive cells (mean ± SD).

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