Journal: bioRxiv

Article Title: Acquisition of cell migration defines NK cell differentiation from hematopoietic stem cell precursors

doi: 10.1101/142380

Figure Lengend Snippet: YTS or NK92 NK cell lines or human NK cells (eNK) were labeled with CellTracker Violet then co-cultured with an irradiated EL08.1D2 monolayer for 24 hours in a 96-well plate. Images were acquired continuously every 2 minutes. A) Representative phase-contrast images of each cell type with randomly selected sample tracks overlaid. Insets show zoomed-in views of single-cell tracks. Scale bar=300 μm. B) Mean track speed (top), straightness (center), and arrest coefficient (bottom) of NK cells were calculated. Error bars indicate s.d. Means with significant differences were determined by ordinary one-way ANOVAwith Tukey’s multiple comparison test **p< 0.01, ****p< 0.0001). Data are representative of three independent experiments. n=75 (NK92), 205 (YTS), 250 (eNK). C) Rose plots of representative tracks. n=30 per graph.

Article Snippet: T and B cell lineage depletion was performed using NK cell RosetteSep (STEMCELL Technologies) and Ficoll-Paque density gradient centrifugation from routine red cell exchange apheresis performed at Texas Children’s Hospital.

Techniques: Labeling, Cell Culture, Irradiation, Comparison

Journal: bioRxiv

Article Title: Acquisition of cell migration defines NK cell differentiation from hematopoietic stem cell precursors

doi: 10.1101/142380

Figure Lengend Snippet: CD34 + HSCs were seeded on a monolayer of EL08.1D2 cells. Cells were imaged continuously in phase-contrast mode for 28 days. FACS analysis was performed weekly to monitor expression of developmental markers. Representative images of NK cell intermediates with randomly selected tracks 0 (i), 7 (ii), and 21 (iii) days after start of experiment. Scale bar=50 mm. B) Mean speed, displacement, and path length of NK cell developmental intermediates were measured at 7-day intervals as indicated. Error bars indicate s.d. ****p< 0.0001 by ordinary one-way ANOVAwith Tukey’s multiple comparisons test. n=932 (0D), 803 (7D), 134 (14D), 148 (21D). C) Mean speed, displacement, and path length of cells from continuous tracking from the first 14 days are shown as 24 hour segments. Error bars indicate s.d. Means with significant differences as analyzed by ordinary one-way ANOVAwith Tukey’s multiple comparison test are shown (*p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001). Sample size for each individual time point are listed in Materials and Methods. D) FACS analysis of NK cell maturation markers. Predicted NK cell developmental stage based on phenotype as described in the text is shown in roman numerals. All data shown are representative of 3 independent experiments.

Article Snippet: T and B cell lineage depletion was performed using NK cell RosetteSep (STEMCELL Technologies) and Ficoll-Paque density gradient centrifugation from routine red cell exchange apheresis performed at Texas Children’s Hospital.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Acquisition of cell migration defines NK cell differentiation from hematopoietic stem cell precursors

doi: 10.1101/142380

Figure Lengend Snippet: A) Straightness and arrest coefficient for NK cell tracks at weekly time points. Error bars indicate s.d. Means with significant differences as analyzed by ordinary one-way ANOVAwith Tukey’s multiple comparison test are shown (**p< 0.01, ***p< 0.001, ****p< 0.0001). n=932 (0D), 803 (7D), 134 (14D), 148 (21D). Straightness and arrest coefficient for NK cell tracks at daily time points. Error bars indicate s.d. Means with significant differences are determined by ordinary one-way ANOVAwith Tukey’s multiple comparison test (*p< 0.05, ***p< 0.001, ****p< 0.0001). Sample size for each individual time point are listed in Materials and Methods. All data shown are representative of 3 independent experiments.

Article Snippet: T and B cell lineage depletion was performed using NK cell RosetteSep (STEMCELL Technologies) and Ficoll-Paque density gradient centrifugation from routine red cell exchange apheresis performed at Texas Children’s Hospital.

Techniques: Comparison

Journal: bioRxiv

Article Title: Acquisition of cell migration defines NK cell differentiation from hematopoietic stem cell precursors

doi: 10.1101/142380

Figure Lengend Snippet: A) Mean square displacement (MSD) of tracks acquired at weekly time points. Graph is truncated at 450 min because few cell tracks persist for longer. Error bars indicate s.d. B) Representative NK cell track after 21 days of development shown with segments corresponding to each migration mode labeled.Fraction of time spent in either constrained, random, or directed motion for each cell after 0 days of development. n=932. D) Fraction of time spent in either constrained, random, or directed motion for each cell after 21 days of development. n=148. All data shown are representative of 3 independent experiments.

Article Snippet: T and B cell lineage depletion was performed using NK cell RosetteSep (STEMCELL Technologies) and Ficoll-Paque density gradient centrifugation from routine red cell exchange apheresis performed at Texas Children’s Hospital.

Techniques: Migration, Labeling

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: Antigen-specific CD4 and CD8 memory cells show dissociated production of IL-2 and IFN-γ. Healthy subject (n = 22) PBMC ( panels A–C ) or CD4 vs. CD8 depleted PBMC ( panel D ) (300,000 cells per well) were challenged in 20 h culture with recall protein (mumps or candida) or peptide (EBV BMLF-1, EBNA3a, or EBNA3b) antigen and IFN-γ/IL-2 producing cell frequency was measured by ELISPOT method. Assays were performed in triplicate. For panels A–C each line represents a separate subject (n = 12). Panel D , CD4 vs. CD8 depletion analysis reveals immune response phenotype. Spot forming units (sfu) observed with PBMC (300,000 cells per well) were assigned as 100%. Sfu observed when plating the same number of CD4 depleted PBMC or CD8 depleted PBMC are shown as a proportion (%) of PBMC activity.

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques: Enzyme-linked Immunospot, Activity Assay

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: Allo-antigen-specific T cells show dissociated IL-2-IFN-γ production. Allogeneic 20 h cultures were performed as 2-way MLR with 3 × 10 5 PBMC ( panels A–C ), or as a 1-way allogeneic response using CD3/CD56 depleted PBMC stimulators (300,000 cells/well) and CD4 ( panels D–F ), or CD8 ( panels G–I ) cell responders (300,000 cells/well). IL-2 sfu ( panels A, D, G ), IFN-γ sfu ( panels B, E, H ) and proliferation cpm ( panels C, F, I ) are shown. Stimulator identification is shown on the x-axis, while responder identification is shown in the legend. Shaded regions highlight discordance between IL-2 and IFN-γ producing effector function. Stimulator or responder cell alone control cultures resulted in <5 sfu and <1,000 cpm (not shown).

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques:

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: Correlations among IFN-γ, IL-2, and proliferation.

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques:

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: The allogeneic proliferative response entails a substantial non-T cell bystander component. Panel A. representative proliferative reaction by CFSE analysis. CFSE labeled allogeneic responder PBMC (300,000 cells/well) were cultured with CD3/56 depleted PBMC for 6 days. Lymphocyte gate was determined by forward and side scatter. CFSE dye dilution was analyzed on responder alone (left panels) vs. responder and stimulator (right panels) gating on CD4, CD8 and CD4-/CD8- cell fractions. Reactions for 4 separate stimulators (represented on x -axis) and responders (represented in legend) are shown in panels B–E , representing CD4 ( panel B ), CD8 ( panel C ), and CD4-/CD8- ( panel D ) proliferation by CFSE dye dilution analysis, or bulk proliferation by 3 H thymidine incorporation ( panel E ).

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques: Labeling, Cell Culture

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: During mixed lymphocyte reactions soluble factors are likely involved with proliferation. Associations between CD4 T cell and CD8 T cell ( panel A ), between CD4 T cell and CD4-/CD8- cell ( panel B ), and between CD8 T cellCD4 T cell and CD4-/CD8- cell ( panel C ) proliferation as determined by CFSE dye dilution method for reactions described in .

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques:

Journal: Cells

Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

doi: 10.3390/cells1020127

Figure Lengend Snippet: Soluble antigen specifc proliferative response also entails a substantial non-T cell bystander component. PBMC from two individuals with candida specific IL-2 and IFN-γ secreting CD4 T cell populations (identified in as CD4 T cell mediated activity) were analyzed by CFSE dye dilution analysis of 6 day antigen specific proliferation reactions. Analysis of CD4, CD8, and CD4-/CD8- cell fractions proliferating in response to soluble antigen are represented for each individual.

Article Snippet: PBMC, CD3- depleted PBMC (>97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC (>95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R&D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

Techniques: Activity Assay