Figure 3.
BHLHE40 is not required for generating allo-antigen specific Tr1 cells. (A) BHLHE40 knockout strategy. 3 sgRNA (red) were designed to simultaneous target the N’ terminus exon 5 of BHLHE40. Protein domains (black) and exons (grey) are annotated. (B) High knockout efficiency in naïve CD4+ T cells. Indels % were calculated 2-3 days after nucleofection. N = 15 healthy donors. (C) Schematic of allo-antigen Tr1 cell induction of sgBHLHE40-edited cells with iDC, DC-10, or control matDC. (D, E) CD4+ T cell characterization after primary MLR with (D) DC-10 or (E) iDC. %Tr1 cells 10 days after primary MLR. Tr1% is measured by flow cytometry and gated on live/CD3+/CD4+/CD45RA-/CD49b+/LAG3+ cells (left). Allo-antigen specific anergy assay of cells restimulated in a secondary MLR with matDC. % anergy of mock-treated and sgBHLHE40-edited (middle). Proliferation response, as indicated by CFSE dye dilution, of mock-treated and sgBHLHE40-edited cells taken at day 10 of the primary MLR after restimulation with Dynabeads for 3 days (right). n = 5 healthy donors.