Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

“…To delineate how CAR activation influences these functionalities compared to endogenous TCR/CD28 activation, we first employed a modified in vitro T-cell suppression assay where Tregs were activated via CAR or TCR/CD28 overnight and then co-incubated with CellTrace dye-labeled CD4 + and CD8 + T responder (Tresp) cells activated with anti-CD3/CD28 beads overnight in parallel at different Treg to Tresp cell ratios 48,49 . Interestingly, CAR-activated Tregs were less efficacious than their TCR/CD28-activated counterparts in inhibiting CD4 + ( Figure 3A ) and CD8 + ( Figure 3B ) Tresp cell proliferation.…”

“…T cells were activated with anti-CD3/CD28 beads (Gibco, ThermoFisher Scientific) at a 1:1 ratio and recombinant human IL-2 (Peprotech, ThermoFisher Scientific), and expanded in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), glutamax, penicillin-streptomycin, HEPES, non-essential amino acids (NEAA), and sodium pyruvate (all from Gibco, ThermoFisher Scientific). Tregs were cultured with 1,000 IU/ml IL-2, CD4 + Teff cells with 100 IU/ml IL-2, and CD8 + T cells with 300 IU/ml IL-2 48 . Antibodies used for FACS and flow cytometry can be found in Table S9 .…”

“…CAR Tregs were activated via CAR (with irradiated CD19-K562 cells), via TCR/CD28 (with irradiated CD64-CD80-K562 cells loaded with anti-CD3 OKT3 antibody) or left resting (with irradiated K562 cells) at a 1:1 Treg to target cell ratio in round bottom 96-well plates. In parallel, CD4 + and CD8 + T responder (Tresp) cells were mixed at a 1:1 ratio, labeled with CellTrace Violet (CTV) or CellTrace Far Red (CTFR) according to the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific), and activated with anti-CD3/CD28 beads at a 1:2 bead to cell ratio overnight 48,90 . The following day, Tresp cells were debeaded and co-incubated with activated Tregs in round bottom 96-well plates at different Treg:Tresp ratios for three days in the absence of exogenous IL-2 48,90 .…”

“…In parallel, CD4 + and CD8 + T responder (Tresp) cells were mixed at a 1:1 ratio, labeled with CellTrace Violet (CTV) or CellTrace Far Red (CTFR) according to the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific), and activated with anti-CD3/CD28 beads at a 1:2 bead to cell ratio overnight 48,90 . The following day, Tresp cells were debeaded and co-incubated with activated Tregs in round bottom 96-well plates at different Treg:Tresp ratios for three days in the absence of exogenous IL-2 48,90 . Co-cultures were then harvested, stained for CD4 and CD8, and CTV or CTFR dye dilution measured via flow cytometry.…”