CCL3 as Possible Negative Prognostic Factor in Chronic Lymphocytic Leukemia.

INTRODUCTION: Both microenvironmental signals from surrounding cells and changes in the genome of leukemic cells play essential role in the development of chronic lymphocytic leukemia. Nurse-like cells (NLCs) are one of the important elements of the microenvironment of CLL cells. The key role in the interactions of leukemic cells with NLCs is played by chemokines, which may interfere with the programmed cell death process in the leukemic lymphocytes. The aim of our study was analysis of selected microenvironmental factors having a potential impact on the leukemic cells survival, as well as their association with clinical, cytogenetic, and molecular parameters. For this study, we selected three types of molecules which can modulate microenvironment: chemokines IL-8 and CCL3 (which are classically secreted to extracellular matrix), soluble forms of adhesion molecules JAG1 and CD163, and secreted form of endogenous protein BIRC5. We assessed their expression in the serum of CLL patients as well as in medium of long-term NLCs cultures. METHODS: Long-term cell culture was prepared from mononuclear cells derived from the blood of 34 patients with CLL. Number of NLCs cells was evaluated, under a light inverted microscope. The concentration of IL-8, CCL3, sBIRC5, sCD163, and sJAG1 in culture medium and serum was assessed by enzyme-linked immunosorbent assays. RESULTS: There were significant differences in the concentration of IL-8, sBIRC5, CCL3, sCD163, and sJAG1 between the patient's blood serum and the culture medium. The concentrations of IL-8, CCL3, and JAG1 were higher in the culture medium, which confirmed the role of the microenvironment in the production of these proteins. In addition, the concentration of CCL3 chemokine in both patient's blood serum and in the culture medium correlated with the number of NLCs and with known prognostic factors in the course of CLL, e.g., Rai stage, WBC, expression of ZAP-70, CD38, and CD5/19. CONCLUSION: The microenvironment of CLL cells, which includes NLCs, plays an important role in the pathogenesis of CLL. The CCL3 chemokine seems to be a good factor representing microenvironment of CLL cells. Chronic lymphocytic leukemia is a complex and very heterogeneous disease; therefore, its progress should be considered both in the context of genetic changes and the interaction with microenvironmental cells.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

Programmed Cell Death-1 and Its Ligands as Targets for Therapy of Multiple Myeloma Patients.

Purpose: Among hematological malignancies, the expression profile of programmed cell death-1 (PD-1) and its ligands in multiple myeloma (MM) is still debated by numerous research groups. In current study, we characterized the expression of PD-1 and its ligands both on RNA and protein levels in MM patients. We have also attempted to analyze whether daratumumab therapy might overcome CD38-mediated immunosuppression that inhibits in particular CD8+ T-cell function. Patients and Methods: This study included 149 newly diagnosed MM patients and 15 relapsed/refractory MM patients before and after daratumumab treatment. The mRNA levels of PDCD1, PDCD1LG1, PDCD1LG2 and their splicing variants was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Flow cytometry was used to characterize the surface expression of PD-1 and its ligands on plasma cells, B and T cells. The surface expression of PD-1 on T cells was assessed by flow cytometry before and after daratumumab treatment. Results: The mRNA expression of PDCD1LG1, PDCD1LG2 and their splicing variants were higher in plasma cells as compared to bone marrow mononuclear cells (BMMCs). Our results show that the percentage of plasma cells expressing PD-L1 was significantly higher than plasma cells expressing PD-L2 (p<0.0001) in bone marrow (BM) of MM patients. There was no significant difference between the percentage of plasma cells expressing PD-1 and B cells expressing PD-1 in BM of MM patients (11.19% vs 8.91%). We also found that the percentage of CD8+PD-1+ T cells was significantly higher than CD4+PD-1+T cells in BM (p<0.0001) of MM patients. Here, we observed no change in PD-1 expression on CD4+ and CD8+ T cells after the daratumumab treatment. Conclusion: The PD-1 and its ligands might represent an interesting target for MM immunotherapy, as one would target both malignant plasma cells as well as the immune cells that play a key role in tumor escape mechanisms.

Prognostic Value of Tie2-Expressing Monocytes in Chronic Lymphocytic Leukemia Patients.

Tie2-expressing monocytes (TEMs) are associated with tumor progression and metastasis. This unique subset of monocytes has been identified as a potential prognostic marker in several solid tumors. However, TEMs remain poorly characterized in hematological cancers, including chronic lymphocytic leukemia (CLL). This study analyzed, for the first time, the clinical significance of TEM population in CLL patients. Flow cytometry analysis of TEMs (defined as CD14+CD16+Tie2+ cells) was performed at the time of diagnosis on peripheral blood mononuclear cells from 104 untreated CLL patients. Our results revealed an expansion of circulating TEM in CLL patients. These monocytes express high levels of VEGF and suppressive IL-10. A high percentage of TEM was associated closely with unfavorable prognostic markers (ZAP-70, CD38, 17p and 11q deletion, and IGHV mutational status). Moreover, increased percentages of circulating TEMs were significantly higher in patients not responding to the first-line therapy as compared to responding patients, suggesting its potential predictive value. High TEM percentage was also correlated with shorter overall survival (OS) and shorter time to treatment (TTT). Importantly, based on multivariate Cox regression analysis, TEM percentage was an independent predictor for TTT. Thus, we can suggest the adverse role of TEMs in CLL.

The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia.

PURPOSE: NOTCH1 mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1 mut allelic burden. This study compared two methods of the NOTCH1 mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients. PATIENTS AND METHODS: This study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann-Whitney, Fisher's exact test, Kruskal-Wallis, Kaplan-Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data. RESULTS: We proved that ddPCR increased the detectability of the NOTCH1 mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1 mut group of patients compared to the NOTCH1 wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHV mut NOTCH1 wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1 mut determined by ddPCR constituted as a dependent prognostic factor for TTFT. CONCLUSION: The selection of the precise method of NOTCH1 mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1 mut irrespective of the quantity of allelic burden.

In vivo, ex vivo and in vitro dasatinib activity in chronic lymphocytic leukemia.

Dasatinib inhibits the breakpoint cluster region-Abelson murine leukemia 1 (BCR-ABL1) gene along with other kinases known to be overexpressed and abnormally active in patients with chronic lymphocytic leukemia (CLL). The current study used primary leukemic cells obtained from 53 patients with CLL that were treated with dasatinib. A 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and Annexin V staining was performed to assess the cytotoxic effects of dasatinib treatment. The XTT assay revealed that the median cytotoxicity of dasatinib was 8.30% (range, 0.00-77.89%). Due to high dispersion of dasatinib activity, patients were divided into sensitive (n=27; 50.94%; median cytotoxicity, 22.81%) and resistant groups (n=26; 49.06%; median cytotoxicity, 0.00%). A median cytotoxicity of 8.30% was selected as a cut off value. Using Annexin V staining and flow cytometry on exemplary sensitive and resistant CLL samples, it was revealed that 17.71 and 1.84% of cells were apoptotic, respectively. The current study presented a case of a patient with concomitant occurrence of CLL and chronic myeloid leukemia (CML) with a major molecular response after dasatinib treatment. A simultaneous reduction of circulating CLL cells indicated in vivo anti-CLL activity induced by dasatinib. After an in vitro culture of the patient's mononuclear cells with subsequent dasatinib treatment, a higher percentage of CLL cells undergoing apoptosis was obsevered when compared with untreated samples (38.19 vs. 21.99%, respectively). Similarly, the percentage of CLL apoptotic cells (ΔΨmlow) measured by chloromethyl-X-rosamine was higher after incubation with dasatinib (7.28%) than in the negative control (2.86%). In conclusion, dasatinib induced antileukemic effects against CML and CLL cells. The results of the current study indicated that dasatinib may induce apoptosis ex vivo, in vitro and in vivo in CLL.

Differential Function of a Novel Population of the CD19+CD24hiCD38hi Bregs in Psoriasis and Multiple Myeloma.

Psoriasis (Ps), an autoimmune disease, and multiple myeloma (MM), a blood neoplasm, are characterized by immune dysregulation resulting from the imbalance between the effector and regulatory cells, including B regulatory (Breg) lymphocytes. Peripheral blood samples from 80 Ps patients, 17 relapsed/refractory MM patients before and after daratumumab (anti-CD38 monoclonal antibody) treatment, 23 healthy volunteers (HVs), and bone marrow samples from 59 MM patients were used in the study. Bregs were determined by flow cytometry using CD19, CD24, and CD38. Intracellular production of interleukin-10 (IL-10) was assessed by flow cytometry after CD40L, LPS, and CpG stimulation. IL-10 serum or plasma concentrations were tested using ELISA method. The percentage of CD19+CD24hiCD38hi Bregs was not different whereas the production of IL-10 in Bregs was significantly higher in Ps patients in comparison with HVs. The percentage of CD19+CD24hiCD38hi Bregs in MM patients was significantly higher than in HVs (p < 0.0001). The percentage of CD19+CD24hiCD38hi Bregs was significantly higher in MM patients with the ISS stage I (p = 0.0233) while IL-10 production in Bregs was significantly higher in ISS stage III (p = 0.0165). IL-10 serum or plasma concentration was significantly higher in Ps and MM patients when compared to HVs (p < 0.0001). Following the treatment with daratumumab the percentages of CD19+CD24hiCD38hi Bregs significantly decreased (p < 0.0003). Here, in the two opposite immune conditions, despite the differences in percentages of Bregs in Ps and MM we have identified some similarities in the IL-10 producing Bregs. Effective treatment of daratumumab besides the anti-myeloma effect was accompanied by the eradication of Bregs.

The Correlation of Mutations and Expressions of Genes within the PI3K/Akt/mTOR Pathway in Breast Cancer-A Preliminary Study.

There is an urgent need to seek new molecular biomarkers helpful in diagnosing and treating breast cancer. In this elaboration, we performed a molecular analysis of mutations and expression of genes within the PI3K/Akt/mTOR pathway in patients with ductal breast cancer of various malignancy levels. We recognized significant correlations between the expression levels of the studied genes. We also performed a bioinformatics analysis of the data available on the international database TCGA and compared them with our own research. Studies on mutations and expression of genes were conducted using High-Resolution Melt PCR (HRM-PCR), Allele-Specific-quantitative PCR (ASP-qPCR), Real-Time PCR molecular methods in a group of women with ductal breast cancer. Bioinformatics analysis was carried out using web source Ualcan and bc-GenExMiner. In the studied group of women, it was observed that the prevalence of mutations in the studied PIK3CA and AKT1 genes was 29.63%. It was stated that the average expression level of the PIK3CA, PIK3R1, PTEN genes in the group of breast cancer patients is lower in comparison to the control group, while the average expression level of the AKT1 and mTOR genes in the studied group was higher in comparison to the control group. It was also indicated that in the group of patients with mutations in the area of the PIK3CA and AKT1 genes, the PIK3CA gene expression level is statistically significantly lower than in the group without mutations. According to our knowledge, we demonstrate, for the first time, that there is a very strong positive correlation between the levels of AKT1 and mTOR gene expression in the case of patients with mutations and without mutations.

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 < 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.

Cofilin-1 Maintains Prosurvival Signaling in Chronic Lymphocytic Leukemia Cells.

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.

Expression and Clinical Significance of Neuropilin-1 in Patients With Multiple Myeloma.

BACKGROUND: Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor A (VEGFA), and has been reported to be overexpressed in several malignancies. Since angiogenesis plays an important role in pathogenesis of multiple myeloma (MM) and the role of NRP1 in MM has not been studied yet, we characterized the expression of NRP1 in this disease. MATERIALS AND METHODS: The expression level of NRP1 was measured in 140 patients newly diagnosed with MM and 28 healthy controls by flow cytometry and quantitative reverse transcriptase polymerase chain reaction. RESULTS: Expression of NRP1 was significantly reduced on plasma cells (median=2.05%) compared to that on B-cells (median=10.05%, p<0.0001) in bone marrow of patients with MM. In MM, the expression of NRP1 was high on plasmacytoid dendritic cells (median=85.85%) and low on regulatory T-cells (median=0.6%). CONCLUSION: In MM, NRP1 is regulated differentially as compared to other B-cell malignancies at both the RNA and protein level.

An association of circulating Tregs and Th17 cells producing IL-21 and IL-22 with the ROMA in ovarian cancer patients.

The purpose of the present study was to assess the association of regulatory T cells (Tregs; CD4+ FOXP3+) and helper T lymphocytes (Th17) releasing interleukin (IL)-21 and IL-22, with the Risk of Ovarian Malignancy Algorithm (ROMA). Similar association was made with two additional tumour markers, human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) from patients serum. The presence of Tregs and Th17 was determined both in the peripheral blood and in the tissue of epithelial ovarian tumors. Mononuclear cells obtained from patient's peripheral blood (PBMCs) and from ovarian tissue were isolated by density gradient centrifugation. As a control group patients who had undergone surgery for infertility without ovarian pathology were selected. The percentage of Tregs and Th17 releasing IL-21 or IL-22 cells from both peripheral blood and tumor tissue was measured by flow cytometry. No differences in demographic parameters like body mass index, age, or gravidity were observed among the studied groups. However, an increased concentration of marker HE4 and value of ROMA was identified in individuals with ovarian cancer when compared with women with cystadenomas. Furthermore, a negative correlation between the ROMA value in the serum and Tregs from the peripheral blood of patients with cystadenoma ovarian tumors was detected. The presented work documents, for the first time, the negative association between peripheral blood Tregs and ROMA evaluation based on the tumour markers present in the serum of women with ovarian cystadenoma. Such an effect might result from the negative impact of Tregs on the inflammation process and on tumorigenesis caused by the persistent inflammation.

The influence of a green roof drainage layer on retention capacity and leakage quality.

The aim of the research was to determine the influence of the substrate and different drainage materials on retention capacity and runoff water quality from three green roof containers. Phosphates were chosen as the water quality indicator based on their potential adverse impact on water quality in urban rainwater collectors. The field experiment was conducted at the Warsaw University of Life Sciences Water Center meteorological station in years 2013-2015. In terms of precipitation, the monitoring period covered a wet (+147.1 mm), average (+42.7 mm) and dry (- 66.3 mm) year. Leakage from the containers was recorded when the substrate moisture exceeded 20% and precipitation exceeded 3.5 mm/d for washed gravel, or 5.0 mm/d for a polypropylene mat and expanded clay. Phosphates were observed in leachates from all containers, with higher values observed in the second year of monitoring. As the result of this study, it can be concluded that the polypropylene mat and aggregates create different conditions for the formation of the leachate, in both volumes and its chemistry. The drainage layer made from a polypropylene mat is the most effective in terms of rainwater retention capacity and the resulting leachate quality.

Prognostic impact of NOTCH1, MYD88, and SF3B1 mutations in Polish patients with chronic lymphocytic leukemia.

INTRODUCTION    Currently available prognostic factors determining the course of chronic lymphocytic leukemia (CLL) are not fully efficient, especially for newly diagnosed patients. Investigation of molecular changes may help clarify the reasons for the heterogeneity of the disease. Apart from already confirmed TP53 mutations, the novel candidates: NOTCH1, SF3B1, and MYD88 might represent clinically relevant biomarkers. OBJECTIVES    The aim of this study was to evaluate the mutational status of NOTCH1, MYD88, and SF3B1 and to compare the results with confirmed prognostic factors: ZAP­70, CD38, and immunoglobulin heavy­chain variable region (IGHV) mutation in CLL. The study assessed also prognostic significance in terms of the time to first treatment (TTFT) and subset analysis. PATIENTS AND METHODS The study was conducted on samples of 370 newly diagnosed patients with CLL. The analysis was performed using high­resolution melting, Sanger sequencing, and polymerase chain reaction methods. RESULTS    Patients harboring the NOTCH1 mutation were significantly more often found among patients with an unmutated IGHV gene status and high expression of CD38 and ZAP­70. The MYD88 mutation was equally distributed in patients with mutated and unmutated IGHV status (5 vs 7 patients). For MYD88 and SF3B1, there were no significant differences in the levels of CD38 and ZAP­70 expression. The tendency for lower median TTFT was revealed in patients with mutated SF3B1 (P = 0.08). The analysis showed the presence of 14 different types of the subsets of IGHV in 50 of 345 patients (14.5%). The most frequent were subsets #1 and #2. CONCLUSIONS    The NOTCH1 and SF3B1 mutations accompany biological markers of unfavorable prognosis in patients with CLL. The mutations may contribute to the identification of patients with high­risk CLL.

Expression of circulating miRNAs associated with lymphocyte differentiation and activation in CLL-another piece in the puzzle.

Expression of microRNAs is altered in cancer. Circulating miRNA level assessed in body fluids commonly reflects their expression in tumor cells. In leukemias, however, both leukemic and nonleukemic cells compose circulating miRNA expression profile of peripheral blood. The latter contribution to extracellular miRNA pool may result in specific microenvironmental signaling, which promotes proliferation and survival. In our study, we used qT-PCR to assay peripheral blood serum of 22 chronic lymphocytic leukemia (CLL) patients for the expression of 84 miRNAs associated with activation and differentiation of B and T lymphocytes. Results were analyzed regarding the most important prognostic factors. We have found that the general expression of examined miRNAs in CLL patients was lower as compared to healthy volunteers. Only miR-34a-5p, miR31-5p, miR-155-5p, miR-150-5p, miR-15a-3p, and miR-29a-3p were expressed on a higher level. Alterations of expression observed in CLL patients involved miRNAs associated both with B and T lymphocyte differentiation and activation. The most important discriminating factors for all functional miRNA groups were trisomy 12, CD38 expression, B2M level, WBC, and NOTCH1 gene mutation. Correlation of expression of miRNAs related to T lymphocytes with prognostic factors proves their supportive function in a leukemic microenvironment. Further studies utilizing a larger test group of patients may warrant the identification of circulating miRNAs that are key players in intercellular interactions and should be considered in the design of microenvironment-targeted therapies.

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 we found CFL1 overexpression and low expression of MYH9 in comparison with healthy volunteers. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 66·7%, VIM in 87·5% and CFL1 in 62·5% CLL patients in an Enzyme-Linked ImmunoSpot assay. Low frequencies of autoreactive peptide-specific T cells were detected against MYHIIA, VIM and CFL1 in CLL patients ex vivo; most of the detected cells had an effector-memory phenotype. Our findings support the existence of cytotoxic immune responses against three autoantigens that have been identified as targets of CLL clonotypic B-cell receptors. The presence of autoreactive CD8(+) T cells against MYHIIA, VIM and CFL1 in CLL patients indicates the involvement of antigen-specific autoreactive T cells in the pathogenesis of CLL.

Accumulation of CD5+CD19+ B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils.

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4+ cells (70.3 %) than on CD8+ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19+ B lymphocytes (6.5 %), while CD5+CD19+ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5+CD19+ B cells (16.5 %) than on CD19+ B cells (3.5 %) and on CD4+ T cells (20 %) than on CD8+ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5+CD19+ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.

Expression of Programmed Death 1 Ligand in Different Compartments of Chronic Lymphocytic Leukemia.

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 PDL1 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.