Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 (BCR method) in the absence (control; CTRL) or presence of TAC (6ng/ml) or SRL (2ng/ml or 6ng/ml) and flow cytometric analyses were performed after 6 days in culture. (A) A representative experiment: cells were gated on viable lymphocytes and analyzed for CFSE diluting proliferating cells. This scheme of analysis was used in all subsequent experiment, unless indicated otherwise. (B) The percentage of proliferating CD19 + B cells as obtained in A from 7 different experiments. (C) Absolute number of proliferating CD19 + B cells was calculated in each experiment by multiplying the recovered cell counts with the percentage of proliferating cells as in A (n = 7). Statistically significant (*p < 0.05) inhibition of B cell proliferation was observed with SRL at both subtherapeutic (2ng/ml) and therapeutic (6ng/ml) doses.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Control, Inhibition

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were stimulated with anti-IgM, anti-CD40 mAb and IL-21 and multi-color flow cytometric analyses were performed on day 6 as described in . The figures A , B and C show the expression of various surface markers on stimulated B cells (Percentage of positive cells/total proliferating CD19 + cells) p < 0.05, **p < 0.01. CTRL, control; TAC6, 6 ng/ml TAC; SRL2, 2 ng/ml SRL; SRL6, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Expressing, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: B cells were purified by depleting non-B cells resulting in >95% CD19 + cells which subsequently sorted into CD27 − (naïve) and CD27 + (memory) B-cell fractions. These subsets were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of TAC or SRL at 6ng/ml and were analyzed by multicolor flow cytometry after 6 days in culture. (A) A representative experiment: histogram plots show dilution of CFSE in the proliferating CD19 + CD27 − (upper panel) or CD19 + CD27 + (lower panel) cells. (B) Data are from four different independent experiments are shown as mean ± SD percent proliferating naïve CD19 + CD27 − and memory CD19 + CD27 + B cells. (C) B cell subsets showing indicated surface markers were analyzed and plotted as mean ± SD (n = 4) percent of proliferating cells in the cultures of naïve CD19 + CD27 − (upper panel) and memory CD19 + CD27 + B cells (lower panel). The residual cells that proliferated in presence of SRL demonstrated an activated phenotype. *p < 0.05, **p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were cultured as in Figs and . (A) A representative experiment showing flow cytometric profile indicative of putative plasma cells (CD19 low ) in proliferated B cells (gated on viable lymphocytes; see ). (B) The mean ± SD percentage of such CD19 low B cells from 4 different independent experiments. (C) Mean ± SD (n = 4) percentage of CD19 low CD38 ++ plasmablasts, CD19 low CD138 + plasma cells, Blimp1 + PAX5 - cells and CD138 + Blimp1 + cells in the proliferating CD19 low cells. *p < 0.05, **p < 0.01. CTRL, control; TAC, 6 ng/ml TAC; SRL, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Clinical Proteomics, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were pre-stimulated for 6 days with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of 6ng/ml TAC or SRL. These cultured B cells were used as stimulators in 6-day MLRs of allogeneic CFSE-labelled CD4 + CD25 − T cell responders. ( A ) Level of proliferation differentially induced by pre-cultured B cells as detected by CFSE dilution in the allogeneic CD4 responder cells (representative experiment on the left, and compiled data from 8 independent experiments on the right). ( B ) Percentage of responding T cells positive for memory marker (CD45RO) and activation markers (CD62L, CD25, CD69, CD95) after co-culture with pre-stimulated B cells (n = 8); ( C ) Mean ± SD (n = 4) percentage of responding proliferating T cells expressing intracellular cytokines (top row) and transcription factors (bottom row). Taken together the data indicated that B cells that proliferated in presence of SRL on a per cell basis were capable of inducing alloreactive T cell proliferation towards a Th1 phenotype. *p < 0.05. ** p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Marker, Activation Assay, Co-Culture Assay, Expressing

Journal: Stem Cell Reports

Article Title: Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

doi: 10.1016/j.stemcr.2015.09.012

Figure Lengend Snippet: In Vivo Generation of Myeloid, B, and T Cells from IdHP Cells (A) Schematic representation of the in vivo model for investigating the developmental potential of IdHP cells. (B) Flow cytometric analysis of cells in the peripheral blood of mice transplanted with control or IdHP cells 4 weeks after injection. Donor derived PBMCs (CD45.1 + ) were analyzed for the expression of MAC1 versus CD19 and CD3 versus NK1.1. (C) Flow cytometric analysis of cells in thymus, spleen, and BM of mice transplanted with IdHP cells 7 weeks after injection. (D) The number of T (CD4 + CD8 + ) cells in thymus, B (IGM + ) cells in spleen, and myeloid (MAC1 + GR1 + ) cells in BM generated from the IdHP cells. (E) CD4 + T cells in spleen generated from the IdHP cells were sorted and labeled with CFSE. The labeled cells were stimulated with plate-coated anti-CD3/28 for 4 days. Flow cytometric analysis of the cells after stimulation is shown (n = 3). ∗∗∗ p < 0.001. Data are shown as mean ± SD from three independent experiments. See also Figure S2 .

Article Snippet: The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119: 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR1 (553128), NK1.1 (553165), TCRβ (553172), human CD11C (347637), human CD25 (555432), human CD33 (555450), allophycocyanin (APC)-conjugated CD45.1 (558701), CD45.2 (558702), C-KIT (553556), CD19 (550992), human CD19 (555415), human CD56 (555518) and human HLA-DR (559866).

Techniques: In Vivo, Injection, Derivative Assay, Expressing, Generated, Labeling

Journal: Stem Cell Reports

Article Title: Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

doi: 10.1016/j.stemcr.2015.09.012

Figure Lengend Snippet: In Vivo Generation of Myeloid, B, and T Cells from BM-Derived IdHP Cells (A) ID3 and control retroviruses were infected with LKS cells in BM of B6CD45.1 mice, and the infected cells were sorted and cultured on TSt-4 stromal cells in the presence of SCF, IL-7, and FLT3-L for 2 weeks. Flow cytometric profiles of CD19 versus B220 gated on MAC1 − GR1 − NK1.1 − cells are shown. (B) BM IdHP cells (1 × 10 6 ) were intravenously injected into sublethally irradiated NOG mice. Flow cytometric analysis of cells in thymus, spleen, and BM of mice transplanted with BM IdHP cells 7 weeks after injection is shown. (C) The number of T (CD4 + CD8 + ) cells in thymus, B (IGM + ) cells in spleen, and myeloid (MAC1 + GR1 + ) cells in BM generated from the IdHP cells (n = 3) is shown. Data are shown as mean ± SD from three independent experiments (n = 3). (D) Id3 and control viruses were infected with HSC (CD34 − LKS), MPP (CD34 + LKS), CLP (LIN − C-KIT + IL7R + ), and CMP (LIN − C-KIT + SCA-1 − CD16/32 − CD34 + ) cells in BM of B6CD45.1 mice, and the infected cells were sorted and cultured on TSt-4 stromal cells in the presence of SCF, IL-7, and FLT3-L for 2 weeks. Flow cytometric profiles of CD19 versus B220 gated on MAC1 − GR1 − NK1.1 − cells are shown. See also Figure S4 .

Article Snippet: The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119: 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR1 (553128), NK1.1 (553165), TCRβ (553172), human CD11C (347637), human CD25 (555432), human CD33 (555450), allophycocyanin (APC)-conjugated CD45.1 (558701), CD45.2 (558702), C-KIT (553556), CD19 (550992), human CD19 (555415), human CD56 (555518) and human HLA-DR (559866).

Techniques: In Vivo, Derivative Assay, Infection, Cell Culture, Injection, Irradiation, Generated

Journal: Stem Cell Reports

Article Title: Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

doi: 10.1016/j.stemcr.2015.09.012

Figure Lengend Snippet: Inducible Generation of IdHP Cells Using an ID3-ER Retrovirus (A) Developmental potential of FL (LKS) progenitors transduced with control (ER) and ID3-ER retrovirus. After transduction, GFP + cells were sorted and cultured on TSt-4 stromal cells supplemented with SCF, IL-7, and FLT3-L in the presence or absence of 4-OHT for 4 weeks. Representative flow cytometric profiles for CD19 versus B220 are shown. (B) Expansion of FL (LKS) progenitors transduced with control and ID3-ER retrovirus in the presence of 4-OHT. Viable cells were counted at the indicated time points. (C) Number of LIN − cells from control and ID3-ER infected cells after 32 days of culture. (D) B cell generation from ID3-ER-transduced cells after withdrawal of 4-OHT. The percentage of CD19 + cells at the indicated time points is shown. ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are shown as mean ± SD from three independent experiments. See also Figure S5 .

Article Snippet: The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119: 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR1 (553128), NK1.1 (553165), TCRβ (553172), human CD11C (347637), human CD25 (555432), human CD33 (555450), allophycocyanin (APC)-conjugated CD45.1 (558701), CD45.2 (558702), C-KIT (553556), CD19 (550992), human CD19 (555415), human CD56 (555518) and human HLA-DR (559866).

Techniques: Transduction, Cell Culture, Infection

Journal: Stem Cell Reports

Article Title: Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

doi: 10.1016/j.stemcr.2015.09.012

Figure Lengend Snippet: Generation of IdHP Cells from Human CB HSCs (A) Flow cytometric analysis of cells derived from CD34 + human CB cells transduced with control and human ID3 retrovirus. After transduction, GFP + cells were sorted and cultured on TSt-4 stromal cells in the presence of SCF, IL-7, FLT3-L, and thrombopoietin (TPO) for 4 weeks. (B) Wright’s stain of hIdHP and control cells. Scale bars, 10 μm. (C) Expansion of hIdHP and control cells on TSt-4 stromal cells in the presence of human SCF, IL-7, FLT3-L, and TPO. Viable cells were counted at the indicated time points (n = 3). (D and E) NK cell (D) and DC (E) generation from hIdHP cells in vitro. Flow cytometric analysis is shown. (F and G) Generation of CD19 + and CD14 + cells from hIdHP cells in NOG mice. (F) Schematic representation of the examination of the developmental potential of hIdHP cells. (G) Flow cytometric profiles for CD33 versus CD19 and CD33 versus CD14 of human CD45 + cell in BM from transplanted mice. Data are shown from three independent experiments. See also Figure S6 .

Article Snippet: The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119: 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR1 (553128), NK1.1 (553165), TCRβ (553172), human CD11C (347637), human CD25 (555432), human CD33 (555450), allophycocyanin (APC)-conjugated CD45.1 (558701), CD45.2 (558702), C-KIT (553556), CD19 (550992), human CD19 (555415), human CD56 (555518) and human HLA-DR (559866).

Techniques: Derivative Assay, Transduction, Cell Culture, Staining, In Vitro

Journal: Cancer discovery

Article Title: Tuning the Antigen Density Requirement for CAR T Cell Activity

doi: 10.1158/2159-8290.CD-19-0945

Figure Lengend Snippet: (a) Primary diagnostic samples of diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) were analyzed by flow cytometry for expression of CD19 compared to normal B cells from healthy donors. Shown is CD19 protein expression, relative to healthy donor PBMC B cells on a Log2 scale. DLBCL: n=8, FL: n=27, CLL: n=13, MCL: n=35. Statistical differences between groups were analyzed by one-way ANOVA non-parametric test with Dunns post-test correction. (b) Representative contour plots illustrating expression levels of CD19 and CD20 in three DLBCL cases as compared to PBMC B cells from healthy donors. (c) Flow cytometric analysis of the expression levels of truncated CD19 on the surface of a library of NALM6 clones. Number of molecules of CD19 for each clone were semiquantitatively determined by the BD Quantibrite kit. (d) NALM6 clones expressing indicated densities of surface CD19 molecules were cocultured at a 1:1 ratio with CD19–4-1BBζ CAR T cells and tumor cell killing was measured in an Incucyte assay. Representative of six experiments with different T cell donors. Statistical analysis performed with repeated measures ANOVA. (e) CD19–4-1BBζ CAR T cells were labeled with cell trace violet (CTV) and then cocultured at a 1:2 ratio with NALM6 clones expressing either 963 or 45,851 molecules of surface CD19. T cell proliferation was measured by flow cytometry four days later. Representative of three experiments with different T cell donors. (f) CD19–4-1BBζ CAR T cells were cocultured with NALM6 clones expressing various amounts of CD19 for 24 hours and secreted IL-2 was measured by ELISA. Shown is the concentration of cytokine measured as compared to log of the CD19 molecule number for that specific clone and curve fitting was done using a four-parameter variable slope dose-response curve. Representative of six experiments with different T cell donors. For all experiments, error bars represent SD. p < 0.05 was considered statistically significant, and p values are denoted with asterisks as follows: p > 0.05, not significant, NS; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Number of molecules of CD19 for each clone were semiquantitatively determined by the BD Quantibrite kit. (d) NALM6 clones expressing indicated densities of surface CD19 molecules were cocultured at a 1:1 ratio with CD19–4-1BBζ CAR T cells and tumor cell killing was measured in an Incucyte assay.

Techniques: Diagnostic Assay, Flow Cytometry, Expressing, Clone Assay, Labeling, Enzyme-linked Immunosorbent Assay, Concentration Assay