Programmed Death-1 and Its Ligand Are Novel Immunotolerant Molecules Expressed on Leukemic B Cells in Chronic Lymphocytic Leukemia

Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies

Aberrant Expression of TLR2, TLR7, TLR9, Splicing Variants of TLR4 and MYD88 in Chronic Lymphocytic Leukemia Patients

Functional toll-like receptors (TLRs) could modulate anti-tumor effects by activating inflammatory cytokines and the cytotoxic T-cells response. However, excessive TLR expression could promote tumor progression, since TLR-induced inflammation might stimulate cancer cells expansion into the microenvironment. Myd88 is involved in activation NF-κB through TLRs downstream signaling, hence in the current study we provided, for the first time, a complex characterization of expression of TLR2, TLR4, TLR7, TLR9, and MYD88 as well as their splicing forms in two distinct compartments of the microenvironment of chronic lymphocytic leukemia (CLL): peripheral blood and bone marrow. We found correlations between MYD88 and TLRs expressions in both compartments, indicating their relevant cooperation in CLL. The MYD88 expression was higher in CLL patients compared to healthy volunteers (HVs) (0.1780 vs. 0.128, p &lt; 0.0001). The TLRs expression was aberrant in CLL compared to HVs. Analysis of survival curves revealed a shorter time to first treatment in the group of patients with low level of TLR4(3) expression compared to high level of TLR4(3) expression in bone marrow (13 months vs. 48 months, p = 0.0207). We suggest that TLRs expression is differentially regulated in CLL but is similarly shared between two distinct compartments of the microenvironment

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

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

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

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