Journal: eLife

Article Title: IFNγ induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation

doi: 10.7554/elife.41641

Figure Lengend Snippet: Figure 2. ASC development from BN precursors is enhanced in Th1 containing co-cultures. Cartoon (a) depicting day 6 paired co-cultures containing Th1 (Be1 co-cultures) or Th2 (Be2 co-cultures) effectors generated from the same HD, BN cells from a second allogeneic HD and exogenous IL-21 and IL-2. Flow cytometric analysis showing T-bet expression (b) on gated HD B cells (non-ASCs) from Be1 and Be2 co-cultures. Phenotyping (c) of day 6 B cell-gated Be1 cells showing T-bet expression in combination with other surface markers. (d–g) ASC development in HD day 6 paired Be1 and Be2 co-cultures showing representative flow plots (d) and frequencies (e) of CD38hiCD27+ ASCs in CD19+/lo-gated B lineage cells. Frequencies of IgM+ (f) or IgG+ (g) ASCs in day 6 paired Be1 and Be2 co-cultures. See Figure 2—figure supplement 1 for BN isolation strategy and characterization of polarized Th1 and Th2 effectors. See Figure 2—figure supplement 2 for gating strategy to identify IgG+ and IgM+ ASCs. See Figure 2—figure supplement 3 for proliferation analysis of B cells in paired day 6 HD Be1 and Be2 co-cultures. Analyses in (b–c) are from representative co-cultures (n > 30). Experiments (e–g) performed on 15 (e), 8 (f) or 6 (g) independent paired Be1 and Be2 co-cultures. Statistical analyses were performed using a non- parametric Wilcoxon paired t test (e) or paired Student’s t test (f–g). P values *<0.05, **<0.01, ****<0.0001. DOI: https://doi.org/10.7554/eLife.41641.005 The following figure supplements are available for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.41641 23 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PercP/Cy5.5 Mouse Anti-Human CD4 (clone OKT4) eBioscience 45-0048-42 (1:200) Antibody BV510 Mouse Anti-Human CD4 (clone OKT4) Biolegend 317444 (1:100) Antibody Fitc Mouse Anti-Human CD11c (clone Bu15) Biolegend 337214 (1:200) Antibody PE Mouse Anti-Human CD11c (clone Bu15) Biolegend 337205 (1:400) Antibody PercP/Cy5.5 Mouse Anti-Human CD14 (clone HCD14) Biolegend 325621 (1:200) Antibody Fitc Mouse Anti-Human CD19 (clone LT19) Miltenyi 302256 (1:100) Antibody PE Mouse Anti-Human CD19 (clone HIB19) Biolegend 302208 (1:200) Antibody PercP/Cy5.5 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302230 (1:100) Antibody APC Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 555415 (1:200) Antibody APC-H7 Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 560727 (1:100) Antibody BV421 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302234 (1:200) Antibody V500 Mouse Anti-Human CD19 (clone HIB19) BD Horizon 561121 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD21 (clone Bu32) Biolegend 354908 (1:100) Antibody Fitc Mouse Anti-Human CD23 (clone M-L23.4) Miltenyi 130-099-365 (1:100) Antibody PE Mouse Anti-Human CD23 (clone EBVCS-5) Biolegend 338507 (1:200) Antibody APC Mouse Anti-Human CD23 (clone M-L233) BD Pharmingen 558690 (1:200) Antibody Fitc Mouse Anti-Human CD27 (clone M-T271) Biolegend 356404 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD27 (clone M-T271) Biolegend 356408 (1:100) Antibody APC Mouse Anti-Human CD27 (clone M-T271) Biolegend 356410 (1:200) Continued on next page Zumaquero et al. eLife 2019;8:e41641.

Techniques: Generated, Expressing, Isolation

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Construction and characterization of the CD19-IgG 1 Fc fusion proteins. A . Diagrams of the lentiviral vector provirus constructs used to transduce the CD19sIg fusion genes. B . Reduced protein electrophoresis by SDS PAGE of fusion protein products after concentration and purification using Protein A Dynabeads. Centricon concentrated supernatant samples from fresh Pro293a™-CDM (A) , 293 T cells (B) , 293 T cells expressing CD19sIg1-3 (C) , and 293 T cells expressing CD19sIg1-4 (D) . Lanes E and F are the DYNAL purified extracts for CD19sIg1-3 and CD19sIg1-4 (respectively). Arrows indicate the predicted size of the monomer, 47 kDa for CD19sIg1-3 and 57 kDa for CD19sIg1-4. All samples were reduced prior to loading. C . Native protein electrophoresis of CD19sIg1-4 after concentration and purification using Protein A Dynabeads; arrow points to 171 kDa band (expected size for trimers of the fusion protein). D . Results of a comparative ELISA using FMC63 monoclonal capture antibody, of purified fusion proteins, in native (N) or denatured (D) forms. E . Results of ELISA using antibodies targeting human CD19 molecule (FMC63, HIB19, F-3 and 2E2B6B10), human CD20 (B9E9) and PSMA (YPSMA-1).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Plasmid Preparation, Construct, Transduction, Protein Electrophoresis, SDS Page, Concentration Assay, Purification, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Evaluation of CD19sIg1-4 fusion protein on primary human cell populations. Flow cytometry plots using FITC-conjugated anti-IgG Fc F(ab’) 2 fragment (FITC-anti-IgG Fc, left panels) or Alexa Fluor 488-labeled CD19sIg1-4 (AF488-CD19sIg1-4, right panels) for detection of 5% anti-CD19 CAR-transduced human primary T-cells mixed with (A) human peripheral blood mononuclear cells (PBMC), (B) NSG bone marrow (NSG BM), and (C) humanized NSG bone marrow ( hu NSG BM).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Flow Cytometry, Labeling

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Evaluation of CD19sIg1-4 fusion protein for detection of anti-CD19 CAR-modified primary human T-cells. Flow cytometry plots demonstrating the sensitivity of detection of anti-CD19 CAR-transduced human primary T-cells mixed in increasing numbers of non-transduced (NT) T-cells using FITC-conjugated anti-IgG Fc F(ab’) 2 fragment (FITC-anti-IgG Fc, upper panels) or Alexa Fluor 488-labeled CD19sIg1-4 (AF488-CD19sIg1-4, lower panels).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Modification, Flow Cytometry, Labeling

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Comparison of CD19sIg1-4 fusion protein to similar commercially available reagents. Flow cytometry plots of staining of primary human T-cells, non-transduced and 5% CAR-transduced, using FITC-conjugated F(ab’) 2 fragment goat anti-human IgG1 Fc γ (A) , biotinylated Protein L (B) , Alexa Fluor 488-labeled CD19sIg1-4 (C) and Alexa Fluor 488-labeled rhCD19-Fc fusion protein (D) . E . Results of staining of the same cell population after pre-incubation of Alexa Fluor 488-labeled CD19sIg1-4 with anti-CD19 monoclonal antibody FMC63.

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Comparison, Flow Cytometry, Staining, Labeling, Incubation

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 1 Chimeric antigen receptor (CAR) detection methods. Binding mechanisms of the different CAR detection reagents. (1) The APC- conjugated AffiniPure F(ab')2 fragment goat antihuman IgG, Fcγ fragment specific, also called anti-Fc, binds the human IgG1 derived hinge domain. (2–4) These three methods use a recombinant human CD19 protein fragment able to bind the anti-CD19 single-chain fragment variable (scFv). The CD19his (2) is fused to a histidine tag that is recognized by an APC-conjugated antihistidine antibody. The CD19bio (3) is fused to a biotin tag that is recognized by an APC-conjugated antibiotin antibody. The CD19-FITC (4) is directly conjugated to the FITC fluorochrome. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Binding Assay, Derivative Assay, Recombinant

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 2 Comparison between different chimeric antigen receptor (CAR) staining methods. (A) Histograms of CARCIK-CD19 cells staining with the anti-Fc antibody in presence of complete medium (RPMI + 10% fetal bovine serum) or after 1 h incubation in human serum at 100%. Negative controls are shown in the first row and represent the unstained samples. (B) Dot plot of CARCIK-CD19 cells detection on the infusion bag product and in the peripheral blood (PB) patient sample with the three CAR detection methods. Negative controls are shown in the first row and represent the samples stained with only the secondary antibody for the indirect methods (CD19his and CD19bio), while the unstained sample for the direct method (CD19-FITC). (C) Stain index for six independent PB samples stained with the three CAR detection methods. Stain index = ((MFI CAR+ population) – (MFI CAR population))/(2 standard deviation of the CAR population). Bars indicate the mean of each group of samples. (D) Comparison of CD19his, CD19bio, CD19-FITC CAR staining methods in six different PB samples from patients treated with CARCIK-CD19 cells. Statistically significant differences are noted in each figure (*p < 0.05; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Staining, Incubation, Standard Deviation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 3 Specificity and sensitivity of the three chimeric antigen receptor (CAR) staining methods using a recombinant human CD19 protein fragment. (A) The specificity was evaluated by staining healthy donor peripheral blood mononuclear cells with the three CAR detection staining reagents to assess background staining. Data are representative of three different donors acquired in one experiment and bars indicate the mean. (B) The sensitivity was evaluated serially diluting CARCIK-CD19+ cells into untransduced CIK cells at six different dilutions (from 100% to 0%). The graph shows mean values and the standard deviation of three independent experiments for each staining method (CD19his, CD19bio, CD19-FITC). The dotted line represents, in both graphs, the lower limit of quantification (LLOQ) for CD19his, identified at 0.1% CAR+

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Recombinant, Standard Deviation

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 4 Comparison between chimeric antigen receptor (CAR) expression measured with CD19his flow cytometry and real-time polymerase chain reaction (PCR) data. (A) CAR detection was concurrently assessed in 56 postinfusion peripheral blood samples from 4 patients treated with CARCIK-CD19 cells by flow cytometry using the CD19his method (CD3+CAR+ cell/μL) and qPCR (VCN/μg of DNA). The results show clear inter-method concordance by Pearson correlation test (R = 0.51, p = 0.0008). Data are displayed in a logarithmic scale to better represent values <1 cell/μL. (B,C) Monitoring of CARCIK-CD19 expansion, using CD19his flow cytometry staining and qPCR, in two representative patients. (D–F) CARCIK-CD19 cells were expanded in vitro and purified on a magnetic column for CAR expression. Data are representative of five independent experiments. Untransduced CIK cells, CAR+ and CAR purified cells were then analyzed by qPCR and for their cytotoxic activity. In each histograms columns represent the mean and bars the standard deviation. (D) CAR gene integration measured with quantitative PCR and expressed as vector copy number/μg of DNA. (E) CAR mRNA expression measured with qPCR and expressed as CAR mRNA copies normalized on the reference gene GUS. (F) Cytotoxic activity against a CD19+ REH cell line at a 1:1 effector to target ratio, after 72 h coculture. Statistically significant differences are noted in each figure (*p < 0.05; **p < 0.01; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining, In Vitro, Purification, Activity Assay, Standard Deviation, Plasmid Preparation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 5 Inclusion of CD19his chimeric antigen receptor (CAR) staining method in a 12 colors flow cytometry panel. Representation of CD3 cells staining in a single patient treated with CARCIK-CD19 cells at different time point after infusion, using viSNE map visualization tool. To interpret high-dimensional single-cell data that were produced by multicolor flow cytometry panel, we used a tool based on the viSNE algorithm [32], which allows visualization of high-dimensional cytometry data on a two-dimensional map at single-cell resolution and preserve the nonlinearity [33]. In the viSNE map, cell position reflects their proximity in high-dimensional space based on the similarity of marker expression. The first column shows the distribution of the four major T-cell populations: CD4+ CAR+ cells (blue), CD4+ CAR cells (orange), CD8+ CAR+ cells (green), and CD8+ CAR cells (red). All the other columns show the expression level of each marker included in the panel, excluding CD45 and CD3 used for the identification of the T cells. D, day; M, month after CARCIK infusion. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Flow Cytometry, Produced, Cytometry, Marker, Expressing

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 6 Application of the CD19his staining method on different commercial chimeric antigen receptor (CAR)-T products and within different sample sources. (A) Representative dot plots of three peripheral blood (PB) samples from patients previously infused with tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®),brexucabtagene autoleucel (Tecartus®) and lisocabtagene maraleucel (Breyanzi®). (B) Representative dot plots of CAR detection on different sample sources: bone marrow (BM), pleural effusion and cerebrospinal fluid (CSF) in patients infused with CARCIK-CD19 cells. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining