Journal: JCI Insight

Article Title: Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells

doi: 10.1172/jci.insight.123672

Figure Lengend Snippet: (A) Schematic diagram of experimental setup and gating strategy for the FlowPRT cell assay. ΔNGFR or CAR Tregs were incubated with a cocktail of single HLA FlowPRA beads for 30 minutes, and bead-CAR Treg interactions were quantified as the loss of beads in a bead singlet gate based on FSC/SSC profile. (B) Binding to HLA-A*02:01–coated beads for each m/hA2-CAR Treg relative to binding of a ΔNGFR Treg control. Statistical significance determined by 1-way ANOVA and Holm-Šídák post hoc test comparing with mA2-CAR; mean ± SEM; **P < 0.01. (C and D) Correlation between the mean of HLA-A*02:01 binding measured by the FlowPRT cell assay and either (C) HLA-A*02:01 tetramer MFI evaluated by flow cytometry or (D) increase in the proportion of CD69+ cells 16 hours after coculture with HLA-A*02:01 versus negative control HLA-A*24:01 K562 cells. (E) Percent binding of each m/hA2-CAR Treg to the indicated HLA-A alleles after normalization to an ΔNGFR Treg control from the same donor. Dotted line represents 2 SDs from the mean of the bead-only control. For a summary of statistical results in E, see Supplemental Table 1. n = 3–6 from at least 3 independent experiments.

Article Snippet: Specifically, we hypothesized that the One Lambda Flow Panel Reactive Antibody (FlowPRA) single Ag beads previously developed to measure serum alloantibody titers ( 35 ), which consist of fluorescently labeled beads coupled to single HLA Ags, could be adapted to measure alloAg-directed CAR Treg specificity.

Techniques: Incubation, Binding Assay, Flow Cytometry, Negative Control