Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

doi: 10.1016/j.xcrm.2022.100614

Figure Lengend Snippet: Generation of Bw6-specific CAR Teffs with pan-primate α-CD3 aAPCs (A) Cartoon of aAPCs (K562s) engineered to express αCD3 CAR and CD86. (B and C) Parental and engineered K562 aAPCs were stained with biotinylated, recombinant human CD3ε protein plus streptavidin-PE (B) or with His-tagged Cynomolgus macaque CD3ε protein plus α-His antibody (C), followed by α-CD86 antibody. (D) Growth curve of Teffs co-cultured with aAPCs expressing pan-primate αCD3 and human CD86, α-CD3/α-CD28-coated beads, or α-CD2/α-CD3/α-CD28-coated beads. Cells were counted every 2 to 3 days and diluted with media. Data are representative of two independent experiments. (E) Schematic of Bw6-specific CAR. ICD, intracellular domain; TM, transmembrane domain; Vh, antibody variable heavy domain; Vl, antibody variable light domain. (F) Cynomolgus macaque T cells were activated with aAPCs and transduced with χHIV lentiviral vectors encoding Bw6-specific CAR or HLAA2-specific CAR and then stained with both HLA-A2 and HLA-B7 (Bw6) tetramers. (G) HLAA2-specific (blue) or Bw6-specific (red) human CAR T cells were incubated with single-antigen FlowPRA beads before analysis on a flow cytometer. Each peak represents beads conjugated to a unique HLA molecule (black, HLA-A molecules; green, Bw6 + HLA-B molecules; purple, Bw6 − HLA-B molecules). Histograms are gated to depict unbound beads, such that a drop in frequency represents binding to CAR T cells. C, control beads. (H) Human CAR Teffs were co-cultured for 5 h with the indicated target before staining with α-TNFα and α-IL-2 antibodies. Data are representative of three independent experiments.

Article Snippet: Cynomolgus CD3 epsilon protein , ACRO Biosystems , Cat#CDE-C5254.

Techniques: Staining, Recombinant, Cell Culture, Expressing, Transduction, Incubation, Flow Cytometry, Binding Assay, Control

Journal: Cell Reports Medicine

Article Title: Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

doi: 10.1016/j.xcrm.2022.100614

Figure Lengend Snippet: Generation and expansion of Cynomolgus macaque CAR Tregs in vitro (A–C) Outline of Treg expansion protocol. Freshly isolated PBMCs from Cynomolgus macaque were stained with CD4, CD25, CD127, and CD45RA antibodies and flow sorted to obtain the top 1% to 2% of CD25 hi CD127 −/lo population (B) that are CD45RA + (C). (D) Following sort, irradiated α-CD3.CD86 aAPCs were co-cultured with Tregs at one aAPC per one Treg. After 48 h, Bw6-specific CAR lentiviral vector was added, and the scheme outlined in Figure 3 A was followed to expand the Bw6-specific CAR Tregs. Cells were counted every 2 to 3 days, and cell growth was graphed, with each line representing 1 of 19 independent sorts. Arrows indicate days of restimulation with irradiated Bw6.86 aAPCs. (E) Tregs were stained with HLA-B7 (Bw6 + ) tetramer upon resting before each restimulation. Displayed is a representative example showing CAR Treg enrichment after antigen-specific restimulation. (F) Summary data from 12 experiments showing CAR Treg enrichment via restimulation. Each line represents one independent sort and expansion. (G) At the end of expansion, cells were stained for FoxP3 and Bw6-specific CAR expression. (H) Summary of 13 experiments showing the percentage of expanded Bw6-specific Tregs expressing FoxP3, CTLA-4, Helios, and Bw6-specific CAR at the conclusion of expansion. Data are represented as mean ± SEM. (I) Genomic DNA collected at the end of cell culture was assessed for methylation of FOXP3 at the Treg-specific demethylated region (TSDR) by bisulfite sequencing. Each row represents one independent product, and each column represents one CpG locus in the TSDR. (J) Summary of average TSDR demethylation across all loci from 18 experiments with six different animals. Each data point is one Treg expansion, and line represents the group mean.

Article Snippet: Cynomolgus CD3 epsilon protein , ACRO Biosystems , Cat#CDE-C5254.

Techniques: In Vitro, Isolation, Staining, Irradiation, Cell Culture, Plasmid Preparation, Expressing, Methylation, Methylation Sequencing

Journal: Cell Reports Medicine

Article Title: Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

doi: 10.1016/j.xcrm.2022.100614

Figure Lengend Snippet: Cynomolgus macaque CAR T cells demonstrate antigen-specific suppressor function (A and B) Bw6- or HLA-A2-specific CAR Tregs were co-cultured with the indicated aAPC for 24 h and then stained for LAP expression (n = 3 independent experiments). (C) Antigen non-specific suppression was measured by co-culture of unlabeled Bw6-specific CAR Tregs or Bw6-specific CAR Teffs with CellTrace Violet (CTV)-labeled allogeneic PBMCs and α-CD3/α-CD28 beads at the indicated PBMC:Treg ratio for 4 to 5 days. (D) Histograms depict proliferation of the CD4 − CD8 + CTV + cells present in the allogenic PBMCs of experiment performed as depicted in (C). (E) Line graph representing mean ± SEM of seven independent experiments performed as in (D). (F and G) Antigen specific suppression was assessed by mixed lymphocyte reaction (MLR). CTV-labeled responder Teffs and CellTrace Far Red (CTFR)-labeled, Bw6-specific CAR T cells were co-cultured with unlabeled, irradiated allogeneic PBMCs from either Bw6 + or Bw6 − animals. (H) Histograms showing proliferation of the CD4 − CD8 + CTV + Teffs in the MLR performed as depicted in (F) and (G). (I) Histograms showing proliferation of CD4 + CTV − CTFR + Bw6-specific Tregs in same MLR wells as (H). MLRs were assessed between seven pairs of animals in two independent experiments, and similar data were obtained in both experiments.

Article Snippet: Cynomolgus CD3 epsilon protein , ACRO Biosystems , Cat#CDE-C5254.

Techniques: Cell Culture, Staining, Expressing, Co-Culture Assay, Labeling, Irradiation

Journal: Cell Reports Medicine

Article Title: Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

doi: 10.1016/j.xcrm.2022.100614

Figure Lengend Snippet: Adoptive transfer of BW6-specific CAR Tregs into Bw6 + NHP resulted in no adverse events (A) Growth of Bw6-specific CAR Tregs from Bw6 + NHP in vitro . Each black line represents one independent expansion of Bw6-specific CAR Tregs from Bw6 − NHP, while red line represents growth of Bw6-specific CAR Tregs from Bw6 + NHP. Arrows indicate days of restimulation with irradiated Bw6.86 aAPCs. (B) Expression of FoxP3 and Helios at the conclusion of manufacture of Bw6-specific CAR Tregs and CAR Teffs grown from Bw6 + animal. (C) Bw6-specific CAR Tregs were co-cultured for 5 days with Carboxyfluorescein succinimidyl ester (CFSE)-labeled allogeneic PBMCs and α-CD3/α-CD28 beads at the indicated PBMC:Treg ratio to assess non-specific suppressor function as in Figure 3 C. Histograms depict proliferation of the CD4 − CD8 + CFSE + cells present in the allogenic PBMCs from Bw6 − animal. (D) Following adoptive transfer of Bw6-specific CAR Tregs and HLA-A2-specific CAR Teffs to autologous Bw6 + recipient, whole blood was stained with HLA-A2 or HLA-B7 (Bw6 + ) tetramer at indicated time points. Dot plots are gated on CD4 + CD8 − FoxP3 + cells.

Article Snippet: Cynomolgus CD3 epsilon protein , ACRO Biosystems , Cat#CDE-C5254.

Techniques: Adoptive Transfer Assay, In Vitro, Irradiation, Expressing, Cell Culture, Labeling, Staining

Journal: Cell Reports Medicine

Article Title: Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

doi: 10.1016/j.xcrm.2022.100614

Figure Lengend Snippet:

Article Snippet: Cynomolgus CD3 epsilon protein , ACRO Biosystems , Cat#CDE-C5254.

Techniques: Recombinant, Activation Assay, Enzyme-linked Immunosorbent Assay