Paulina Wlasiuk

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.

The significance of soluble HLA-G plasma levels as well as messenger HLA-G for B-cell chronic lymphocytic leukemia (B-CLL)

The immunosuppression accompanies B-cell chronic lymphocytic leukemia (B-CLL) but might be also responsible for disease progression by enabling CLL cells to escape from the immunosurveillance. Some particles involved in the regulation of an immune system might represent prognostic value for B-CLL. Recently we found no correlation between HLA-G on messenger and protein level, suggesting that HLA-G is released in soluble form. To confront this hypothesis we characterized soluble HLA-G (sHLA-G) by the prognostic factors in the first cohort of 34 CLL patients. No correlation was observed between sHLA-G levels in ZAP-70(+) and ZAP-70(-) CLL as well as in CD38(+) CLL and CD38(-) CLL patients. Next, we wondered whether gene expression of HLA-G, which represent the whole HLA-G pool in the cell, posses prognostic value for CLL. In the second cohort of 41 CLL patients we assessed messenger levels of HLA-G by the strongest prognostic factors in CLL including cytogenetics, IgVH mutational status, ZAP-70 as well as CD38. No changes of HLA-G expression levels were found in different CLL groups characterized by IgVH gene mutational status, ZAP-70 as well as CD38. We observed no differences in expression of HLA-G in various cytogenetic groups of CLL including del17p, del13q, del11q, +8q, +3q, del14q and del6q when compared to those with normal karyotype or with 12+. Both, mRNA expression of HLA-G and levels of its soluble form in plasma bring no additional prognostic value for B-CLL patients.

Peptide vaccination induces profound changes in the immune system in patients with B-cell chronic lymphocytic leukemia

Although the immune status of chronic lymphocytic leukemia (CLL) patients is mostly characterized by immunosuppression, there is an accumulation of in vivo (graft-versus-leukemia effect) and in vitro (spontaneous remissions after infections) data that indicates that CLL might be effectively targeted by T-cell based immunotherapy. Recently, we characterized receptor for hyaluronic acid mediated motility (RHAMM) as a preferential target for immunotherapy of CLL. We also completed a RHAMM-derived peptide vaccination phase I/II clinical trial in CLL. Here, we present a detailed immunological analysis of six CLL patients vaccinated with HLA-A2 restricted RHAMM-derived epitope R3 (ILSLELMKL). Beside effective induction of R3-specific cytotoxic T-cells, peptide vaccination caused profound changes in different T-cell subsets as well as cytokines. We present longitudinal analyses of Th17, CD8(+)CD103(+), CD8(+)CD137(+) and IL-17 producing CD8(+) T cells (CD8(+)IL- -17(+)) as well as important cytokines involved in regulation of immune response such as TGF-β, IL-10, IL-2 and TNF throughout the peptide vaccination period.

Expression of soluble HLA-G in multiple myeloma patients and patients with renal failure

Human lymphocyte antigen-G (HLA-G) is an immunosuppressive molecule that induces functional silencing of immune component cells and can be responsible for immunosuppression in patients with multiple myeloma (MM). Immune dysfunction is an important feature of MM and leads to infections as well as may promote disease progression. Ninety-five patients were included in this study. In MM, the sHLA-G levels were increased when compared to healthy volunteers and the levels of sHLA-G correlated with concentration of creatinine. Interestingly, we detected high levels of sHLA-G in patients with renal insufficiency without any malignant disease but levels were lower than in MM.

Total expression of HLA-G and TLR-9 in chronic lymphocytic leukemia patients.

Suppressed immune status facilitates immune escape mechanisms that allow chronic lymphocytic leukemia cells to proliferate and expand. The expression of HLA-G could effectively inhibit the immune response. In immune response inhibitory signals follow activation of immune system which might be occur during bacterial or viral infection in CLL patients. In the current study we characterized two components of immune system, inhibitory molecule HLA-G with its receptor - CD85j and Toll-like receptor 9. The material was obtained from 41 CLL patients and 41 HV with similar median age. In CLL patients expression of intracellular and surface HLA-G and soluble HLA-G levels were significantly higher than in HV. We found higher expression of CD85j compared to HV and the positive correlation between expression of HLA-G and CD85j. All the CLL cells expressed TLR-9, and the level of expression positively correlated with expression of HLA-G and CD85j. Patients with higher expression of intracellular expression of TLR-9 have significantly longer treatment-free survival than patients with low expression of TLR-9 (57 months vs. 8 months, respectively). Summarizing in CLL we characterized activatory and inhibitory components of immune system that might be connected functionally. Analysis of TLR-9 expression might have additional prognostic value for CLL patients.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = − 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

Specific cytotoxic T-cell immune responses against autoantigens recognized by chronic lymphocytic leukaemia cells

Mounting evidence suggests that autoreactivity and inflammatory processes are involved in the pathogenesis of chronic lymphocytic leukaemia (CLL). Cytoskeletal proteins, including non‐muscle myosin heavy chain IIA (MYHIIA), vimentin (VIM) and cofilin‐1 (CFL1), exposed on the surface of apoptotic cells have been identified as autoantigens that are recognized by the specific B‐cell receptors of the CLL cells. In 212 CLL patients analysed with quantitative reverse transcriptase‐polymerase chain reaction

PD1/PD1L pathway, HLA-G and T regulatory cells as new markers of immunosuppression in cancers.

The appropriate function of the immune system depends on the effective regulation of the immune response on multiple levels. The key element of an effective immune response to antigenic stimulation is maintaining a homeostasis between activation and inhibitory function of immunocompetent cells and molecules. In pathological conditions such as chronic infections, autoimmune diseases or cancer there are significant alterations, and prevalence of signals of one type over another. Main markers of these dysfunctions are altered expressions of molecules, such as programmed death-1 (PD-1), Human Leukocyte Antigen G (HLA-G), or changed percentages of T regulatory cells (Treg). These indicators of immune system dysfunction may contribute to disease progression, but also could represent good targets for treatment. Interestingly, in recent years there are many new, interesting reports which showed that the role of PD-1, HLA-G or Treg is ambiguous and not always their higher expression or frequency lead to the progression of disease. Recent studies have shown that Treg can suppress bacteria-driven inflammation which promotes carcinogenesis and thus protect the host from cancer development. Moreover, proliferation of hematological tumor cells expressing ILT-2 receptor can be inhibited by HLA-G, in contrast to solid tumors where HLA-G favors tumor escape. In this paper we present characteristics of expressions of PD-1 and its ligands, HLA-G, and frequency of Treg cells in a variety of physiological and pathological conditions associated with chronic infections, autoimmune diseases and cancer. The understanding of the complex interactions between the functional elements of immune system is essential for a detailed characteristics of the mechanisms leading to the development of diseases and identification of more effective targeted therapies.

Indirect induction of regulatory T cells accompanies immune responses during peptide vaccination of chronic lymphocytic leukaemia patients.

Keywords: chronic lymphocytic leukaemia; thalidomide; T regulatory cells; interleukin 2; peptide vaccination