Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Gating strategy. a For the bone marrow CX3CR1 screening assay, BMMCs were gated on the lymphocyte subset based on scatter properties, subsequently dead cells were excluded, CD3+ cells selected and CD8+, and CD8− cells were gated based on expression of CD8. Finally, expression of CX3CR1 was assessed for the CD3+, CD3+CD8+ and CD3+CD8− subsets. b In the cytotoxicity assay, assessment of peripheral blood lymphocyte effectors was performed by primarily gating on lymphocytes based on scatter properties (right-arrow), and then, unwanted events were excluded using a dump channel designed to remove CD19+ (B cells), CD14+ (monocytes) and dead cells. Subsequently, T cells and NK cells were gated based on expression of CD3 and CD56, (T cells CD3+ and NK cells CD3−CD56+). CD3+ cells were subdivided into CD57+ and CD57− cells. Degranulation (CD107a) and production of TNF and IFNγ were then assessed from PBLs cultured alone or in the presence of target cells (inside black frame). For the T cells, graphs from samples cultured alone (above dotted line) and in the presence of P815 target cells with anti-CD3 (below dotted line) are shown. Analysis of granule content (granzymes and perforin), co-stimulatory receptors and markers of activation were investigated for the same subsets, but only for unstimulated PBLs. Isotype controls were only used for granzyme A, granzyme B and perforin. Investigation of doublet formation between target cells and lymphocytes was performed by first gating on target cells based on scatter properties (down-arrow), then doublets were selected (high FSC-A/FSC-H), and conjugates were defined based on the expression of lymphocyte markers (CD3+, CD4+, CD8+ and CD3+CD57+). c In the cytotoxicity assay, P815 target cell cytotoxicity was assessed by first gating on the target cells based on scatter properties; subsequently, the target cells were selected based on the target cell tracer dye TFL4 (in the APC channel) at the same time gating away cells dead prior to co-culture (NFL1 detected in the Pacific Orange channel), before target cell cytotoxicity was assessed by PanToxiLux fluorescence (converted by granzyme B/caspase-3 present in the target cell from a non-fluorescent substrate into a fluorescent product detectable in the FITC channel). Target cell cytotoxicity; d MDS-L target cells were initially gated out based on light scatter properties; subsequently, CD34 was used to identify the cells and at the same time gating away cells dead prior to initiation of the co-culture (NFL1). CD34 was chosen to identify the target cells as staining by the target cell tracer TFL4 was poor (data not shown). Finally, the MDS-L-directed cytotoxicity was investigated using PanToxiLux, as described for (c). e BMMC CD34+ target cells were gated out as the other target cells based on light scatter properties, TFL4/NFL1, and subsequently cells positive for CD45 and CD33 were gated away (selection of double-negative cells), penultimately CD34+ cells were selected and finally cytotoxicity assessed by PanToxiLux fluorescence as previously described

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Screening Assay, Expressing, Cytotoxicity Assay, Cell Culture, Activation Assay, Co-Culture Assay, Fluorescence, Staining, Selection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Analysis of TCR-induced redirected cytotoxicity and CD3+CD57+ T cell characteristics. Peripheral blood lymphocytes and P815 target cells with added anti-CD3 were co-cultured for 4 h, and target cell cytotoxicity was measured indirectly by flow cytometry using the PanToxiLux assay (OncoImmunin) for the assessment of intracellular granzyme B and caspase 3 activation in target cells. a Scatter plot showing P815 cytotoxicity following co-culture with PBMCs from healthy controls, low- and high-risk MDS patients (white, gray and black circles, respectively) (Kruskal–Wallis with Dunn’s posttest, N = 27, p < 0.05). b Scatter plot showing doublets of P815 cells and CD3+ cells as a percentage of all P815 cells following co-culture with PBMCs from healthy controls, low- and high-risk MDS patients (white, gray and black circles, respectively). (Kruskal–Wallis with Dunn’s post test. N = 27 *p < 0.05, **p < 0.01). c Effector cell degranulation and cytokine production by CD3+CD57+ T cells were measured in parallel co-cultures. Unstimulated CD3+CD57+ T cells were investigated for cytotoxic granule content (granzyme A, granzyme B and perforin), co-stimulatory receptors (DNAM-1, NKG2D and 2B4), co-inhibitory receptors (PD-1 and TIM3), adhesion molecules (CD11a, CD18 and ICAM) and extracellular markers of activation (HLA-DR, CD38, and CD45RA). Bars represent median and interquartile range (K–W with Dunn’s posttest, N = 24, *p < 0.05)

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Cell Culture, Flow Cytometry, Activation Assay, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Reactivity of PBLs toward autologous lymphocyte-depleted bone marrow. a Comparison of degranulation and cytokine production of CD57− and CD57+ T cells as well as NK cells following co-culture of PBMCs with autologous lymphocyte-depleted BMMCs. b Degranulation and production of TNF and IFNγ by CD57+ T cells from low-risk and high-risk MDS patients (gray and black bars, respectively). Bars represent median and interquartile range. c CD34+ BMMC-directed cytotoxicity was measured in co-culture of PBMCs and autologous lymphocyte-depleted BMMCs using the PanToxiLux assay (intracellular granzyme B and caspase 3 activation). No significant difference was observed when comparing PanToxiLux levels in CD34+ cells from low- and high-risk MDS patients (N = 11, M–W) (*p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Comparison, Co-Culture Assay, Activation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Levels of bone marrow CD57+ T cells, CD57+ T cell degranulation and CD34+ BMMC-directed cytotoxicity following initiation of hypomethylating therapy. Assay of serial bone marrow samples taken prior to and following initiation of azacytidine therapy (samples 2 and 3 taken approximately 3–6 months after initiation of treatment). a Frequency of CD57+ T cells in bone marrow samples from the three time points. b Degranulation of CD57+ peripheral blood T cells collected prior to treatment with azacytidine when co-cultured with autologous T and NK cell-depleted BMMC samples from the given time points. c Expression of PD-1 on CD57+ T cells from bone marrow samples collected prior to and following treatment with azacytidine. d CD34+ BMMC-directed cytotoxicity using the PanToxiLux assay (granzyme B and caspase-3 activation) for co-cultures PBMCs collected prior to azacytidine treatment with autologous lymphocyte-depleted BMMCs from the given time points. Symbols used (diamond, square, circle, up- and down-facing triangles) correspond to individual patients in figures a–c

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Cell Culture, Expressing, Activation Assay