Median expression of PD-1 and PD-L1 splicing variants.

<p>The expression levels of PD-1, PD-L1 and their splicing variants were calculated as an inverse ratio of the difference in cycle threshold (ΔCt) method, where ΔCt is the Ct value of the target splicing variant minus the Ct value of GAPDH.</p

PD-1 expression after CLL cells stimulation with CD40L and IL-4.

<p>Figure displays analysis of PD-1 expression on both transcript and protein levels on CLL cells after stimulation with CD40L and IL-4. (A) PD-1 mRNA expression level in stimulated cells and non-stimulated control (0.22 vs. 0.24, p = 0.54). (B) PD-1 MFI in stimulated and non-stimulated cells (82.22 vs. 69.34, p = 0.48). (C) Difference of PD-1 mRNA expression levels between stimulated cells and non-stimulated control assessed using qRT-PCR (2^−ΔΔCt) and correlated with CD38 MFI, with samples segregated into up-regulation or down-regulation of CD38 upon stimulation (0.637 vs. 0.326; p = 0.34). (D) Change of PD-1 MFI between stimulated and non-stimulated cells, with patients segregated as in (C) (6.240 vs. −6.670, p = 0.0093).</p

Schematic representation of the organization of PD-1 (A) and PD-L1 (B) splicing variants.

<p>Primer localization is marked with arrows. Primers were designed to anneal at exon junctions that are specific for the particular splicing variant.</p

Primer sequences.

<p>F, forward; R, reverse; fl_PD-1, full length PD-1; Δex2_PD-1, PD-1 lacking exon 2; Δex3_PD-1; PD-1 lacking exon 3; Δex2,3_PD-1, PD-1 lacking exons 2 and 3; Δex2,3,4_PD-1, PD-1 lacking exons 2, 3 and 4; fl_PD-L1, full length PD-L1; Δex2_PD-L1, PD-L1 lacking exon 2.</p

Surface expression of PD-1 and PD-L1 on cells from CLL patients and HVs.

<p>Figure displays a flow cytometric analysis of PD-1 and PD-L1 expression on CLL cells and normal B cells. (A) Median PD-1 expression of PD-1 on CD5⁺CD19⁺ CLL cells and control B cells of healthy volunteers (HVs) (47.2% vs. 14.81%, p<0.0001). (B) The mean fluorescence intensity MFI of PD-1 on CD5⁺CD19⁺ CLL cells and control B cells of HVs (12.49 vs. 8.59, respectively, p = 0.078). (C) Median PD-L1 expression on CD5⁺CD19⁺ CLL cells and control B cells of HVs (median: 52.52%, range 10.8%–97.3%, p = 0.22). (D) PD-L1 MFI of CD5⁺CD19⁺ CLL cells and control B cells of HVs (9.96 vs. 7.93, p = 0.017). (E) Correlation of MFI of PD-1 positive CLL cells with PD-L1 positive CLL cells (r² = 0.34, p<0.05). MFI of PD-1 and PD-L1 on CD5⁺CD19⁺ leukemic cells and on CD19⁺ control B cells was defined by flow cytometric analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035178#pone.0035178.s002" target="_blank">Figure S2</a>).</p

Clinical characteristics of CLL patients of groups A and B.

<p>A – the group of 43 patients analyzed by qRT-PCR for PD-1, PD-L1 and their splicing variants.</p><p>B – the group of 45 patients analyzed by flow cytometry method.</p><p>Detailed characteristics of CLL patients are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035178#pone.0035178.s004" target="_blank">Table S1</a>.</p