Figure 1
Figure 1. T-cell responses to influenza A vaccination in patients with CML on TKI and healthy controls. PBMCs collected before and 2 to 3 months postvaccination were thawed and stimulated for 24 hours with or without seasonal influenza vaccine at a final concentration of 1.5 µg/mL of hemagglutinin antigens or with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (2 μg/mL; Sigma Aldrich, Gillingham, United Kingdom) (positive control) for 19 hours at 37°C. Brefeldin A (10 µg/mL) (Sigma Aldrich) was added alone or with monensin (0.7 µL/mL) (BD/Pharmingen, San Diego, CA) and the degranulation marker CD107a-FITC (BD/Pharmingen). PBMCs were washed and stained with anti-CD3 and anti-CD8 antibodies, fixed/permeabilized (all BD Biosciences, Oxford, United Kingdom), and stained with anti–IFN-γ, anti–TNF-α, and anti–IL-2 antibodies (all BD/Pharmingen). Data acquisition was performed using FACSCalibur (BD Biosciences, Oxford, United Kingdom), and a minimum of 300 000 events were acquired. The threshold of positivity for cytokines and CD107a was set in order to minimize nonspecific staining in nonstimulated cells (negative control). Following vaccination, a response was considered positive if there was a minimum of 0.10% flu-specific T cells producing TNF-α or INF-γ and the percentage of antigen-specific T cells producing TNF-α or INF-γ was twofold or higher compared with prevaccination level. (A) Examples of preexisting CD8+ and CD4+ T-cell responses to influenza before vaccination in patients on TKI and a healthy control. (B) Examples of T-cell responses to influenza A vaccination in patients on TKI using IC assay. (C) Detection of influenza-specific CD8+ T cells using an HLA-A2–restricted GILGFVFTL (FluMP) pentamer: the fluorescence-activated cell sorter plot from a CML patient on dasatinib showing a robust CD8+ T-cell response to influenza vaccination is presented. Unstimulated PBMCs from HLA-A0201 patients and healthy controls were stained with HLA-A0201/GILGFVFTL (FluMP) Pro5 MHC I Pentamer (ProImmune, Oxford, United Kingdom) conjugated to APC and costained with anti–CD8-FITC (ProImmune) and anti–CD3-PerCP (BD Biosciences).

T-cell responses to influenza A vaccination in patients with CML on TKI and healthy controls. PBMCs collected before and 2 to 3 months postvaccination were thawed and stimulated for 24 hours with or without seasonal influenza vaccine at a final concentration of 1.5 µg/mL of hemagglutinin antigens or with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (2 μg/mL; Sigma Aldrich, Gillingham, United Kingdom) (positive control) for 19 hours at 37°C. Brefeldin A (10 µg/mL) (Sigma Aldrich) was added alone or with monensin (0.7 µL/mL) (BD/Pharmingen, San Diego, CA) and the degranulation marker CD107a-FITC (BD/Pharmingen). PBMCs were washed and stained with anti-CD3 and anti-CD8 antibodies, fixed/permeabilized (all BD Biosciences, Oxford, United Kingdom), and stained with anti–IFN-γ, anti–TNF-α, and anti–IL-2 antibodies (all BD/Pharmingen). Data acquisition was performed using FACSCalibur (BD Biosciences, Oxford, United Kingdom), and a minimum of 300 000 events were acquired. The threshold of positivity for cytokines and CD107a was set in order to minimize nonspecific staining in nonstimulated cells (negative control). Following vaccination, a response was considered positive if there was a minimum of 0.10% flu-specific T cells producing TNF-α or INF-γ and the percentage of antigen-specific T cells producing TNF-α or INF-γ was twofold or higher compared with prevaccination level. (A) Examples of preexisting CD8+ and CD4+ T-cell responses to influenza before vaccination in patients on TKI and a healthy control. (B) Examples of T-cell responses to influenza A vaccination in patients on TKI using IC assay. (C) Detection of influenza-specific CD8+ T cells using an HLA-A2–restricted GILGFVFTL (FluMP) pentamer: the fluorescence-activated cell sorter plot from a CML patient on dasatinib showing a robust CD8+ T-cell response to influenza vaccination is presented. Unstimulated PBMCs from HLA-A0201 patients and healthy controls were stained with HLA-A0201/GILGFVFTL (FluMP) Pro5 MHC I Pentamer (ProImmune, Oxford, United Kingdom) conjugated to APC and costained with anti–CD8-FITC (ProImmune) and anti–CD3-PerCP (BD Biosciences).

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