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

Evaluation of seasonal changes of triterpenic acid contents in Viscum album from different host trees

Context: Viscum album L. (Loranthaceae) is a semi-parasitic plant used in pharmacy and medicine mostly for its hypotensive and anticancer activity. The effects may be related to the presence of triterpenic acids, such as betulinic (BA) and oleanolic (OA) acids. Objectives: In our investigations the content of triterpenic acids in V. album from different host trees depending on the season of harvest was determined. Material and methods: V. album herb was dried and extracted with ethyl acetate using ultrasound energy. The reversed phase HPLC-PDA method was used for the analysis of triterpenic acids. The structure of the target components was confirmed by mass spectrometry with an electrospray ionization source. Results: Diversity in the content of both compounds was noted; however, OA was the dominant triterpenic acid and the amount thereof was ∼10 times higher than that of BA. The analysis of changes in the amount of triterpenic acids during the spring-winter period revealed the highest content of OA in summer (from 6.84 to 13.65 mg/g). In turn, in the other seasons of harvest, the content was in the range of 4.41–9.83, 6.41–9.56 and 5.59–12.16 mg/g for spring, autumn and winter, respectively. In most cases, a similar tendency was observed for BA. Discussion and conclusion: In most cases, the highest amount of the investigated compounds was found in summer; thus, this period seems to be optimal for acquisition of plant material rich in triterpenic acids

In Vitro Antiproliferative Activity of Extracts of Carlina acaulis subsp. caulescens and Carlina acanthifolia subsp. utzka

Various species of the Carlina genus have been used in traditional medicine in many countries to treat numerous skin disorders, including cancer. The objective of this work was to assess the anticancer properties of root and leaf extracts from Carlina acaulis subsp. caulescens and C. acanthifolia subsp. utzka. Anti-tumor properties of the extracts were explored using a tetrazolium-based cell viability assay and flow cytometric apoptosis analysis, followed by immunodetection of phosphoactive ERK1/2 in UACC-903, C32, and UACC-647 human melanoma cell lines. Normal human fibroblasts were used as a control. Leaf extracts inhibited the viability of all tested melanoma cell lines in a dose-dependent fashion while the fibroblasts were less sensitive to such extract. The root extracts inhibited the proliferation of UACC-903 and UACC-647 cells only at the highest doses (300 μg/mL). However, the C32 and fibroblast cells exhibited an increase in the cellular proliferation rate and no caspase activity was observed in response to the root extracts (100 μg/mL). An increase in caspase activity was observed in melanoma cells treated with the leaf extracts of both Carlina species. Leaf extracts from C. acaulis subsp. caulescens (100 μg/mL) inhibited proliferatory ERK1/2 in UACC-903 and C32 cells, as demonstrated by the decrease in ERK1/2 phosphorylation. No reduction in phospho-ERK1/2 was observed in the tested cell lines treated with the root extracts, apart from UACC-647 after incubation with the C. acanthifolia subsp. utzka root extract (100 μg/mL). There was no change in ERK1/2 phosphorylation in the fibroblasts. The extracts from the leaves and roots were analyzed by HPLC and the analysis showed the presence of triterpenes and phenolic acids as the main extract components. The research demonstrated that the extracts from the leaves of the plants were cytotoxic against the human melanoma line and induced apoptosis of the cells. The triterpene fraction present in the tested extracts may be responsible for this activity

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

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

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