Figure 4.
Effect of Kdm1a depletion on components of the CLL microenvironment. (A) iKdm1aKD;Eμ-TCL1A leukemic mice (red) with reduced absolute numbers of CD3+ T cells in PB (top, Dox-treatment for 4 weeks, P = .028) and spleen (bottom, Dox-treatment until end of survival experiment, P = .032) compared with those of Eμ-TCL1A animals (blue, Mann Whitney test). “N” in panels A-E indicates the number of analyzed animals. (B) Representative images of B220 (green)/CD3 (red) immunofluorescence stainings illustrating reduced density of CD3+ T cells in splenic germinal centers of iKdm1aKD;Eμ-TCL1A (bottom) vs Eμ-TCL1A (top) mice (2 weeks of Dox-treatment). (C) Tissue cytometer analysis (StrataQuest) of CD3/KDM1A immunofluorescence stainings of spleen sections. Left: quantification of CD3/KDM1A signals demonstrating decreased CD3+ T cells (P = .029) and confirming KDM1A knockdown in the reduced T cells (P = .038) in spleens of iKdm1aKD;Eμ-TCL1A (red) vs Eμ-TCL1A mice (blue, Mann Whitney test, mean ± SD). Right: representative tissue cytometry plots of CD3/KDM1A stainings. (D) Significantly higher percentage of CD11b+F4/80+ monocytes/macrophages in PB (P = .001) and spleens (P = .002) of iKdm1aKD;Eμ-TCL1A (red) mice vs control (blue) animals (flow cytometry, Mann Whitney test at end point). (E) Left: quantification of immunofluorescence signals from F4/80 and KDM1A costained spleen sections display an increase of F4/80+ macrophages (P = .03) with reduced KDM1A signal in F4/80+ macrophages (P = .01) in iKdm1aKD;Eμ-TCL1A (red) vs Eμ-TCL1A (blue, Mann Whitney test, mean ± SD) mice after 2 weeks of Dox-treatment. Middle: representative tissue cytometry plots of F4/80 and KDM1A costainings. Right: representative immunofluorescence staining of F4/80 (green)/KDM1A (red) in spleen sections from Eμ-TCL1A (top) and iKdm1aKD;Eμ-TCL1A (bottom) mice. (F) Murine primary CLL cells (Eμ-TCL1A with or without induced KDM1A-KD) were cocultured for 48 hours with murine BM stromal cells (BMSCs) supplemented with CpG and IL-15. Flow cytometry for annexin V demonstrates lower viabilities of iKdm1aKD;Eμ-TCL1A leukemic cells cocultured with Eμ-TCL1A BMSCs or with iKdm1aKD;Eμ-TCL1A BMSCs and of Eμ-TCL1A leukemic cells cocultured with iKdm1aKD;Eμ-TCL1A BMSCs (all compared with Eμ-TCL1A leukemic cells cocultured with Eμ-TCL1A BMSCs; all P < .05, Mann Whitney test). (G) Human primary CLL cells as suspension cultures or cocultured with HS-5-shCtrl. or HS-5-shKDM1A stromal cells (confirmed KDM1A-KD in supplemental Figure 3E). Reduced prosurvival effect of HS-5-shKDM1A stromal feeders on CLL cells (annexin V flow cytometry) at 48 hours (P = .012) and 72 hours (P = .004; Student t test, black connecting lines for samples from identical patients). (H) Primary CLL cells cocultured for 48 hours with differentiated THP-1-shCtrl. or THP-1-shKDM1A monocytic cells (confirmed KDM1A-KD in supplemental Figure 3F) supplemented with CpG and IL-15. Top: knockdown of KDM1A in THP-1 cells reduces the percentage of proliferating (Ki-67+) CLL cells (P = .004, Student t test; black connecting lines for samples of identical patients). Bottom: representative histogram of flow-cytometric Ki-67 expression.

Effect of Kdm1a depletion on components of the CLL microenvironment. (A) iKdm1aKD;Eμ-TCL1A leukemic mice (red) with reduced absolute numbers of CD3+ T cells in PB (top, Dox-treatment for 4 weeks, P = .028) and spleen (bottom, Dox-treatment until end of survival experiment, P = .032) compared with those of Eμ-TCL1A animals (blue, Mann Whitney test). “N” in panels A-E indicates the number of analyzed animals. (B) Representative images of B220 (green)/CD3 (red) immunofluorescence stainings illustrating reduced density of CD3+ T cells in splenic germinal centers of iKdm1aKD;Eμ-TCL1A (bottom) vs Eμ-TCL1A (top) mice (2 weeks of Dox-treatment). (C) Tissue cytometer analysis (StrataQuest) of CD3/KDM1A immunofluorescence stainings of spleen sections. Left: quantification of CD3/KDM1A signals demonstrating decreased CD3+ T cells (P = .029) and confirming KDM1A knockdown in the reduced T cells (P = .038) in spleens of iKdm1aKD;Eμ-TCL1A (red) vs Eμ-TCL1A mice (blue, Mann Whitney test, mean ± SD). Right: representative tissue cytometry plots of CD3/KDM1A stainings. (D) Significantly higher percentage of CD11b+F4/80+ monocytes/macrophages in PB (P = .001) and spleens (P = .002) of iKdm1aKD;Eμ-TCL1A (red) mice vs control (blue) animals (flow cytometry, Mann Whitney test at end point). (E) Left: quantification of immunofluorescence signals from F4/80 and KDM1A costained spleen sections display an increase of F4/80+ macrophages (P = .03) with reduced KDM1A signal in F4/80+ macrophages (P = .01) in iKdm1aKD;Eμ-TCL1A (red) vs Eμ-TCL1A (blue, Mann Whitney test, mean ± SD) mice after 2 weeks of Dox-treatment. Middle: representative tissue cytometry plots of F4/80 and KDM1A costainings. Right: representative immunofluorescence staining of F4/80 (green)/KDM1A (red) in spleen sections from Eμ-TCL1A (top) and iKdm1aKD;Eμ-TCL1A (bottom) mice. (F) Murine primary CLL cells (Eμ-TCL1A with or without induced KDM1A-KD) were cocultured for 48 hours with murine BM stromal cells (BMSCs) supplemented with CpG and IL-15. Flow cytometry for annexin V demonstrates lower viabilities of iKdm1aKD;Eμ-TCL1A leukemic cells cocultured with Eμ-TCL1A BMSCs or with iKdm1aKD;Eμ-TCL1A BMSCs and of Eμ-TCL1A leukemic cells cocultured with iKdm1aKD;Eμ-TCL1A BMSCs (all compared with Eμ-TCL1A leukemic cells cocultured with Eμ-TCL1A BMSCs; all P < .05, Mann Whitney test). (G) Human primary CLL cells as suspension cultures or cocultured with HS-5-shCtrl. or HS-5-shKDM1A stromal cells (confirmed KDM1A-KD in supplemental Figure 3E). Reduced prosurvival effect of HS-5-shKDM1A stromal feeders on CLL cells (annexin V flow cytometry) at 48 hours (P = .012) and 72 hours (P = .004; Student t test, black connecting lines for samples from identical patients). (H) Primary CLL cells cocultured for 48 hours with differentiated THP-1-shCtrl. or THP-1-shKDM1A monocytic cells (confirmed KDM1A-KD in supplemental Figure 3F) supplemented with CpG and IL-15. Top: knockdown of KDM1A in THP-1 cells reduces the percentage of proliferating (Ki-67+) CLL cells (P = .004, Student t test; black connecting lines for samples of identical patients). Bottom: representative histogram of flow-cytometric Ki-67 expression.

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