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.

Th17 cells, a recently discovered subset of T helper cells that secrete IL-17A, can affect the inflammation process autoimmune and cancer diseases development. The purpose of this study was to evaluate the role of Th17 cells and IL17A in biology of CLL. The study group included 294 untreated CLL patients in different clinical stages. Here, we show that higher Th17 and IL-17A values were associated with less advanced clinical stage of CLL. Th17 cells’ percentages in PB were lower in patients who died due to CLL during follow-up due to CLL (as compared to surviving patients) and in patients responding to first-line therapy with fludarabine-based regimens (as compared to non-responders). IL-17A inversely correlated with the time from CLL diagnosis to the start of therapy and was lower in patients who required treatment during follow-up. Th-17 and IL-17A values were lower in patients with adverse prognostic factors (17p and 11q deletion, CD38 and ZAP-70 expression). CLL patients with detectable IL-17A mRNA in T cells were in Rai Stage 0 and negative for both ZAP-70 and CD38 expression. Th17 percentages positively correlated with iNKT and adversely with Treg cells. The results of this study suggest that Th17 may play a beneficial role in CLL immunity.

Background Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double‐blind, crossover, confirmatory phase III trial (adept^™2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti‐drug antibodies (ADAs) to vatreptacog alfa. Objectives To characterize the formation of anti‐vatreptacog alfa ADAs in hemophilia patients with inhibitors. Methods/patients This was a post hoc analysis of adept^™2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross‐reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. Results Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow‐up evaluation, the rFVIIa cross‐reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. Conclusions Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Background: The programmed death 1 (PD-1) receptor pathway is responsible for the negative regulation of both T and B lymphocytes upon activation of these cells. There is growing evidence that chronic lymphocytic leukemia (CLL) cells exploit the PD-1 ligand (PD-L1) to resist antitumor immune reactions and maintain their survival by shaping their own microenvironment. Methods: We used a quantitative RT-PCR method to analyze PD-L1 gene expression in bone marrow and peripheral blood mononuclear cells, representing the proliferation and accumulation compartments of CLL. Results: PD-L1 expression was found to be significantly higher in 112 CLL patients than in controls. Levels of PD-L1 expression in bone marrow and peripheral blood were comparable and showed a positive correlation. Furthermore, expression of PD-L1 strongly correlated with expression of PD-1 receptor in mononuclear cells from the same compartment, and was not affected by incubation with immunomodulatory drug thalidomide. Conclusion: PD-L1 expression is shared between CLL cells localized in distinct disease compartments, demonstrating that PD-1/PD-L1 a universal target for therapy.

Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.

In the current study, we analysed the role and prognostic value of myeloid-derived suppressor cells (MDSC) in chronic lymphocytic leukaemia (CLL). The frequency of circulating monocytic MDSC (M-MDSC; defined as CD14⁺CD11b⁺CD15^-HLA-DR^-/low cells) was assessed in correlation with clinical and laboratory parameters characterising the disease activity and patient immune status. Samples of peripheral blood from untreated CLL patients and healthy volunteers were stained with monoclonal antibodies for flow cytometry analysis. CLL patients with M-MDSC percentages above 9.35% (according to the receiver operating characteristic (ROC) analysis) had a shorter time-to-treatment and shorter survival time than the group with a lower percentage of M-MDSC. The M-MDSC percentage was higher in patients with adverse prognostic factors (i.e., 17p and 11q deletion and CD38 and ZAP-70 expression). A high M-MDSC percentage was linked to significantly lower expression of the CD3ζ in T cells. Furthermore, an analysis of immune regulatory molecules (arginase 1 (ARG1), nitric oxide synthase (NOS2), indoleamine 2,3-dioxygenase (IDO), transforming growth factor beta (TGF-β), and interleukin (IL)-10) was performed. By the means of flow cytometry and RT-qPCR, we showed an overexpression of three of them in M-MDSC of CLL patients. M-MDSC cells seem to be an important factor in the immunosuppressive microenvironment of CLL and seem to be a good and novel prognostic factor