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Th17/IL-17A might play a protective role in chronic lymphocytic leukemia immunity.

Hus I, Bojarska-Junak A, Chocholska S, Tomczak W, Woś J, Dmoszyńska A, Roliński J - PLoS ONE (2013)

Bottom Line: Here, we show that higher Th17 and IL-17A values were associated with less advanced clinical stage of CLL.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland.

ABSTRACT
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.

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Flow cytometric analysis of Th17 cells.The dot plots show representative data from healthy control subjects, illustrating the analysis method for identification of CD4/CD3+/IL-17A+ cells (Th17). An acquisition gate was established basing on FSC and SSC that included mononuclear cells. A region, R1, was drawn around the lymphocytes (A). Next, the R1 gated events were analyzed for CD3 PE-Cy5 and CD4 FITC staining and positive cells (CD4+/CD3+) were gated (region R2) (B). The final dot plots CD4FITC versus mouse IgG1 PE (C) and CD4FITC versus IL-17A PE (D) were established by combined gating of events using R1 and R2. The number in the upper right quadrant on the dot plot D represents the percentage of CD4+/CD3+/IL-17A+ (Th17) cells.
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pone-0078091-g001: Flow cytometric analysis of Th17 cells.The dot plots show representative data from healthy control subjects, illustrating the analysis method for identification of CD4/CD3+/IL-17A+ cells (Th17). An acquisition gate was established basing on FSC and SSC that included mononuclear cells. A region, R1, was drawn around the lymphocytes (A). Next, the R1 gated events were analyzed for CD3 PE-Cy5 and CD4 FITC staining and positive cells (CD4+/CD3+) were gated (region R2) (B). The final dot plots CD4FITC versus mouse IgG1 PE (C) and CD4FITC versus IL-17A PE (D) were established by combined gating of events using R1 and R2. The number in the upper right quadrant on the dot plot D represents the percentage of CD4+/CD3+/IL-17A+ (Th17) cells.

Mentions: Intracellular IL-17A analysis was performed on fresh PB samples from 150 CLL patients and 45 HV. Additionally, intracellular IL-17A expression was examined in BM samples from 40 CLL patients. Mononuclear cells (2×106/ml) were cultured in RPMI 1640 supplemented with 2 mmol/L L-glutamine, 5% human albumin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were stimulated with 25 ng/ml of PMA and 1 µg/ml of ionomycin (Sigma, Germany) in the presence of BD GolgiStop (BD Pharmingen, USA) for 5 hours at 37°C in a 5% CO2 atmosphere. Cultured cells were washed twice in PBS, divided into tubes, and then stained with monoclonal antibodies (MoAb) against the cell-surface markers CD4 FITC and CD3 PE-Cy5 (BD Pharmingen). Following membrane staining, cells were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Pharmingen) according to the manufacturer’s instructions. Cells were then intracellularly stained with PE conjugated anti-IL-17A antibodies (BD Pharmingen) or a PE IgG1 isotype control. Finally, cells were washed and analyzed by flow cytometry, performed on a BD FACSCalibur System. Five data parameters were acquired and stored: linear forward and side scatter (FSC, SSC), log FL-1 (FITC), log FL-2 (PE), and log FL-3 (PE-Cy5). For each analysis, 20,000 events were acquired and analyzed using CellQuest Pro software. Isotype-matched antibodies were used to verify the staining specificity and as a guide for setting the markers to delineate positive and negative populations. Dot plots, illustrating the analysis method for the identification of CD4+/CD3+/IL-17A+ (Th17) cells are shown in Figure 1.


Th17/IL-17A might play a protective role in chronic lymphocytic leukemia immunity.

Hus I, Bojarska-Junak A, Chocholska S, Tomczak W, Woś J, Dmoszyńska A, Roliński J - PLoS ONE (2013)

Flow cytometric analysis of Th17 cells.The dot plots show representative data from healthy control subjects, illustrating the analysis method for identification of CD4/CD3+/IL-17A+ cells (Th17). An acquisition gate was established basing on FSC and SSC that included mononuclear cells. A region, R1, was drawn around the lymphocytes (A). Next, the R1 gated events were analyzed for CD3 PE-Cy5 and CD4 FITC staining and positive cells (CD4+/CD3+) were gated (region R2) (B). The final dot plots CD4FITC versus mouse IgG1 PE (C) and CD4FITC versus IL-17A PE (D) were established by combined gating of events using R1 and R2. The number in the upper right quadrant on the dot plot D represents the percentage of CD4+/CD3+/IL-17A+ (Th17) cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815235&req=5

pone-0078091-g001: Flow cytometric analysis of Th17 cells.The dot plots show representative data from healthy control subjects, illustrating the analysis method for identification of CD4/CD3+/IL-17A+ cells (Th17). An acquisition gate was established basing on FSC and SSC that included mononuclear cells. A region, R1, was drawn around the lymphocytes (A). Next, the R1 gated events were analyzed for CD3 PE-Cy5 and CD4 FITC staining and positive cells (CD4+/CD3+) were gated (region R2) (B). The final dot plots CD4FITC versus mouse IgG1 PE (C) and CD4FITC versus IL-17A PE (D) were established by combined gating of events using R1 and R2. The number in the upper right quadrant on the dot plot D represents the percentage of CD4+/CD3+/IL-17A+ (Th17) cells.
Mentions: Intracellular IL-17A analysis was performed on fresh PB samples from 150 CLL patients and 45 HV. Additionally, intracellular IL-17A expression was examined in BM samples from 40 CLL patients. Mononuclear cells (2×106/ml) were cultured in RPMI 1640 supplemented with 2 mmol/L L-glutamine, 5% human albumin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were stimulated with 25 ng/ml of PMA and 1 µg/ml of ionomycin (Sigma, Germany) in the presence of BD GolgiStop (BD Pharmingen, USA) for 5 hours at 37°C in a 5% CO2 atmosphere. Cultured cells were washed twice in PBS, divided into tubes, and then stained with monoclonal antibodies (MoAb) against the cell-surface markers CD4 FITC and CD3 PE-Cy5 (BD Pharmingen). Following membrane staining, cells were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Pharmingen) according to the manufacturer’s instructions. Cells were then intracellularly stained with PE conjugated anti-IL-17A antibodies (BD Pharmingen) or a PE IgG1 isotype control. Finally, cells were washed and analyzed by flow cytometry, performed on a BD FACSCalibur System. Five data parameters were acquired and stored: linear forward and side scatter (FSC, SSC), log FL-1 (FITC), log FL-2 (PE), and log FL-3 (PE-Cy5). For each analysis, 20,000 events were acquired and analyzed using CellQuest Pro software. Isotype-matched antibodies were used to verify the staining specificity and as a guide for setting the markers to delineate positive and negative populations. Dot plots, illustrating the analysis method for the identification of CD4+/CD3+/IL-17A+ (Th17) cells are shown in Figure 1.

Bottom Line: Here, we show that higher Th17 and IL-17A values were associated with less advanced clinical stage of CLL.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus