Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: (A) ATM, 53BP1, and MDC1 protein levels were examined in d4 lysates of ODN ± IL-15-stimulated B-CLL cultures by electrophoretic separation and Western blotting. The asterisk by CLL430 denotes this clone’s lack of ATM protein, due to del11q22 and a coding region mutation (8). MDC1 protein is manifest both as full-length MDC1 protein (~ 225 kDa) and a MDC1 cleavage fragment (~ 70 kDa) (70). Values below each lane represent relative densitometric levels adjusted on the basis of β-actin loading control. (B) Calculated values for CLL430 and CLL515 expression of ATM, 53BP1, and MDC1 (full-length or cleavage fragment) proteins in ODN+IL-15 lysates, as a percent of that seen in ODN-only lysates. (C) Fluorescence histograms representing ATM and 53BP1 protein expression in viable-gated U-CLL1953 cells stimulated with ODN (t=0) and IL-15 (t=20) and harvested at t=68, 92, or 134h. Inserted values represent RMFI (ratio of MFI in test mAb-stained cells (solid line) / MFI in isotype control cells (filled grey). (D) Bar graph of results from the U-CLL1953 experiment showing ATM and 53BP1 protein expression at the intervals following IL-15 pulse to cultures pre-stimulated with ODN for 20h. Data are plotted as a ratio of RMFI in ODN+IL15-treated cells / RMFI in ODN-treated cells (mean ± SEM of staining replicates). (E) Pooled results from CFSE-labeled B-CLL experiments monitoring pSTAT5, ATM protein, and 53BP1 protein levels as a function of division status within 5–6 day cultures stimulated with ODN+IL15 or ODN alone. (Experiments involved n=7 CLL (770, 791, 827, 1031, 1953, 1993, 2018), except for ATM analyses (n=6 CLL). Data expressed as ratio of specific fluorescence in ODN+IL15 cultures versus ODN-only cultures. Dotted line represents normalized fluorescence in ODN-only cultures. Statistical analyses by 2-sided, unpaired t-test: * indicates P=0.02 when compared to ODN only cells; ** indicates P < 0.0001. (G) Linear regression analysis of mRNA versus protein expression of ATM (left) and 53BP1 (right) within 5 B-CLL clones. Specific mRNA was assessed in 20h ODN-primed B-CLL ± additional 20 h of culture with medium or IL-15; specific protein assessed by staining and flow cytometry of day 5–6 cultures stimulated by ODN ± IL-15 (M-1031, M-2018, M-1993, U-1953, & U-1692). mRNA and protein levels are expressed as a ratio of assessed levels in ODN+IL15 cultures versus ODN only cultures.

Article Snippet: For ATM and MDC1: primary mAbs were validated anti-ATM mAb (clone 2C1; Gentex, Irvine, CA) and anti-MDC1 (clone P2B11; LS Bio, Seattle, WA); or IgG1 control (MOPC-21; BD Pharmingen); second stage detection Ab: RPE-conjugated goat F(ab’)2 anti-mouse IgG (H+L), human Ig adsorbed (Southern Biotech, Birmingham, AL).

Techniques: Western Blot, Clone Assay, Mutagenesis, Control, Expressing, Fluorescence, Staining, Labeling, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: Total mRNA was isolated from B-CLL cells that received 20h ODN priming and were subsequently pulsed with IL-15, or medium, for varying intervals. Quantitative RT-PCR with specific primers was performed as detailed in Materials & Methods. IL-15-induced changes in specific mRNA are shown on bar-graph ordinates as fold-increase (ratio of mRNA within ODN+IL15-treated versus ODN only-treated B-CLL), as calculated from ΔCt values by the 2^(-ΔΔCt) method (61). P values for statistical significance between IL-15-pulsed versus non-pulsed cultures were determined by 2-sided, paired t-test. (A) IL-15 influence on MYC and CCND2 mRNA. 4 h pulse = mean of 5 clones tested (CLL 693, 849, 1031, 1692, 1953); 9–16 h = mean of 3 clones tested (CLL 693, 887, 1953); 20–24 h = mean of 5 clones (693, 849, 1031, 1692, 1953). The asterisk linked to NS (not significant) for MYC mRNA indicates that when ΔCt values were used for comparisons, differences +/− IL-15 reached statistical significance (Supplementary Figure 3). (B) IL-15 influence on ATM, P53BP1, and MDC1 mRNA. Levels were significantly reduced upon IL-15 exposure (P<0.001 to 0.003), albeit the suppressive effect on MDC1 mRNA appeared to be transient. 4 h pulse = mean of 6 clones tested (CLL 693, 849, 887, 1031, 1692, 1953); 9–16 h = mean of 3 clones tested (CLL 693, 887, 1953); 20–24 h = mean of 7 clones tested (CLL 693, 849, 887, 1031, 1692, 1993, 2018). (C) Levels of ATM, TP53BP1 and MDC1 mRNA within U-CLL430 and U-CLL515 cells examined in unstimulated state (t=0) or after 4 days of culture with ODN alone or ODN+IL-15. For each mRNA species, mean ΔCt values from triplicate qRT-PCR assays of t=0 unstimulated cells, or d4 cultures stimulated with ODN+IL15, are expressed as a ratio of the mean ΔCt values from d4 cultures exposed to ODN alone, using the 2^(-ΔΔCt) method. Values for U-CLL430 mRNA in ODN and ODN+IL15 cultures represents mean ± SEM values from 3 separate experiments. Asterisks linked to the U-CLL430 experiments indicate that differences in mRNA levels between ODN+IL15 versus ODN only cultures reached statistical significance by 2-sided, paired t-test. Values for U-CLL515 are from one experiment with triplicate qRT-PCR determinations.

Article Snippet: For ATM and MDC1: primary mAbs were validated anti-ATM mAb (clone 2C1; Gentex, Irvine, CA) and anti-MDC1 (clone P2B11; LS Bio, Seattle, WA); or IgG1 control (MOPC-21; BD Pharmingen); second stage detection Ab: RPE-conjugated goat F(ab’)2 anti-mouse IgG (H+L), human Ig adsorbed (Southern Biotech, Birmingham, AL).

Techniques: Isolation, Quantitative RT-PCR, Clone Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: ENCODE Chip Seq data (materials and methods) was used for our bioinformatics evaluation of ATM, TP53BP1, and MDC1 loci. This data base provides information on DNA binding of 161 TFs, including STATs, TBP (TATA-box binding TF) and CTCF (insulator) within several established cell lines, but we here focused on STAT1, STAT3, STAT5, TBP and CTCF binding to promoter regions. Note that ENCODE provides data only for STAT5A binding; binding of closely related STAT5B which shares the same GAS specificity is not indicated. Browser tracks from which this schematic was derived are shown in Supplementary Figure 4.

Article Snippet: For ATM and MDC1: primary mAbs were validated anti-ATM mAb (clone 2C1; Gentex, Irvine, CA) and anti-MDC1 (clone P2B11; LS Bio, Seattle, WA); or IgG1 control (MOPC-21; BD Pharmingen); second stage detection Ab: RPE-conjugated goat F(ab’)2 anti-mouse IgG (H+L), human Ig adsorbed (Southern Biotech, Birmingham, AL).

Techniques: ChIP-sequencing, Binding Assay, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: B-CLL receiving TLR-9 signals, subsequent to BCR internalization of CpG DNA-bearing microbes and/or apoptotic cell debris, upregulate CD122 mRNA/protein and thereby more effectively receive signals from IL-15/IL-15Rα complexes present on adjacent IL-15-producing stromal cells (8, 11, 17) or, alternatively, from soluble IL-15/IL-15Rα complexes cleaved from the latter (128). Earlier studies showed that ODN+IL15-driven in vitro B-CLL growth is positively influenced by del11q22 + del13q14; ATM mutations, and Trisomy 12 (8) and negatively influenced by CD122- or IL-15-specific neutralizing mAbs (17). The present study reveals that inhibitors of JAK1/3, PI-3K, and STAT5 can each block ODN+IL15-driven B-CLL growth, at least in part due to interference with IL-15-driven upregulation of cyclin D2 and IL-15-mediated repression of ATM and 53BP1. (While less clear, it seems likely that pSTAT5 also participates in IL-15-mediated repression of MDC1). Thus, treatment of B-CLL patients with agents that block IL-15 access to its signaling receptor and/or block early activation of JAK, PI-3K, or STAT5 following IL-15/CD122/γc engagement could be effective new approaches to curtailing B-CLL growth within lymphatic tissue of patients.

Article Snippet: For ATM and MDC1: primary mAbs were validated anti-ATM mAb (clone 2C1; Gentex, Irvine, CA) and anti-MDC1 (clone P2B11; LS Bio, Seattle, WA); or IgG1 control (MOPC-21; BD Pharmingen); second stage detection Ab: RPE-conjugated goat F(ab’)2 anti-mouse IgG (H+L), human Ig adsorbed (Southern Biotech, Birmingham, AL).

Techniques: In Vitro, Blocking Assay, Activation Assay