Prognostic significance of CD8 T cells in CLL.

<p>Kaplan-Meier curves were used to analyze the prognostic significance of the relative numbers of CD8. The relative number of CD8 T cells were analyzed by calculating the ratio between the numbers of CD8 T cells and Monoclonal B Clone (MBC) (T/NK cells: MBC ratio) as previously described (4). Patients with relative CD8 T cell count >0.03 (green line) showed significantly higher time to treatment than those with lower numbers of CD8 T cells (blue line). Statistical differences were analyzed by long-rank test.</p

qRT-PCR primers used and thermal profiles.

<p>qRT-PCR primers used and thermal profiles.</p

Absolute numbers of immune cells at diagnosis of CLL.

<p>Absolute numbers of B cells (CD19+), CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+) and NK cells (CD3-CD56+) at diagnosis of 99 CLL patients were compared with 50 healthy controls. Horizontal bars, boxes and whiskers represent median, 25%/75% quartiles and range, respectively. (***p<0.001).</p

Effect of the disease progression on the absolute numbers of B cells, CD4 T cells, CD8 T cells and NK cells.

<p>(A and B) Absolute numbers of B cells, CD8 T cells, NK cells and CD4 T cells were re-evaluated after the evolution of the disease, and they were compared with the values at diagnosis. The median time since the patients were diagnosed was 277 weeks. (C) Absolute NK cell number at the follow-up evaluation was compared with healthy controls. (D and E) Comparison of the absolute numbers of CD8 T cells and NK cells at the follow-up evaluation, according to the chemotherapy treatment. (**p<0.01; ***p<0.001).</p

Clinical characteristics of CLL patients.

<p>MBC: monoclonal B-cells clone.</p><p>* median and range.</p><p>** Positive (>30%).</p><p>Clinical characteristics of CLL patients.</p

Association between clinical parameters of CLL patients and the expression of NKG2D.

<p>NKG2D expression on NK cells was analyzed in patients stratified by the existence of stable and progressive disease (A), Binet stage (B) and Rai stage (C). The percentage of NKG2D+CD4+ T cells in CLL patients was analyzed according to the Binet stage. (**p<0.01).</p

Expression of NKG2D on immune cells of CLL patients.

<p>(A) PBMCs obtained from CLL patients were stained with CD3-, CD4-, CD8-, CD56- and NKG2D-conjugated antibodies. Flow cytometry analysis of one representative patient is shown. (B) The figure shows the comparison of the MFI of NKG2D surface expression in NK cells, CD8 T cells and NKT-like cells between patients and controls. (C) Comparison of the percentage of NKG2D+CD4 T cells between patients and controls. (**p<0.01; ***p<0.001).</p

MSK and DIG-MSK modulate the expression of key angiogenic genes in ovarian carcinoma cells.

<p>a) qRT-PCR analysis of the expression of several key angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared with untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p<0.05; **p<0.01; Mann-Whitney U test). b) Venn diagrams representing genes down-regulated by treatment with MSK or DIG-MSK (p<0.05). Numbers inside the intersections correspond to genes repressed by both treatments.</p

Effect of MTA and DIG-MSK on the secretion of VEGF and the surface expression of VEGFR1 and VEGFR2.

<p>a) Soluble VEGF levels were measured by ELISA in supernatants of ovarian carcinoma cells (A2780, OVCAR-3 and IGROV-1) treated with 200 nM MTA or DIG-MSK compared with untreated cells. b) The surface expression of VEGFR1 and VEGFR2 was analyzed by flow cytometry in ECs treated with 200 nM MTA or DIG-MSK relative to DMSO treated cells. Data represent the mean ± SEM of levels obtained from at least three independent experiments. (*p<0.05; Mann-Whitney U test).</p

Analysis of the expression of key anti-angiogenic genes in ECs treated with MTA or DIG-MSK.

<p>qRT-PCR analysis of the expression of several key anti-angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared to untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate in HUVEC (a) or HMEC-1 (b) endothelial cells. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p<0.05; Mann-Whitney U test).</p