Fig 2: CAR Treg growth and flow phenotype(A–C) Expected CAR Treg growth (A) and expression of FoxP3, CTLA-4, Helios, and CAR on final CAR Treg product (B and C). Each line in panel A represents a single expansion of Tregs, and each arrow represents the day of restimulation with irradiated K562.Bw6.86 cells. Panels B and C represent data from day 18–21 of expansion.

Fig 3: Bw6-specific CAR Tregs traffic to Bw6+ allograft in the bone marrow(A) Outline of transplant. On post-operative day (POD) −8, a Bw6− recipient NHP was rendered diabetic by a single dose of streptozotocin (STZ). Four days later, a first dose of Tregs was administered split between the bone marrow and i.v. Donor islets were harvested on POD −1 and co-cultured with Tregs overnight in media with IL-2. The following day, 1.3 × 109 Tregs were added to the islet/Treg mixture and infused into the bone marrow along with 1.3 × 109 Tregs infused i.v. On POD 4 and POD 14, tertiary and quaternary Treg doses were infused i.v. The islet recipient was given IL-2, rapamycin, and valganciclovir as indicated. Plots are gated on CD4+ CD8− cells.(B) Peripheral blood was stained with Bw6 tetramer to detect Bw6-specific CAR Tregs at the indicated time points.(C) Percent of CD4+ CD8− peripheral blood T cells that expressed FoxP3. Arrows represent infusions of Tregs.(D) Ipsilateral (graft site) or contralateral (non-islet-containing) bone marrow aspirates were stained with Bw6 tetramer at the indicated time points. Plots are gated on CD4+ CD8− cells.(E and F) Flow cytometry phenotype of FoxP3− CAR− (orange), FoxP3+ CAR− (blue), and FoxP3+ CAR+ cells (red). (F) MFI of data represented in (E) is shown.

Fig 4: 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 3C. 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.

Fig 5: CAR Tregs persist and home to bone marrow following adoptive transfer into antigen-negative host(A) Following adoptive transfer of Bw6-specific CAR Tregs into a non-preconditioned, antigen-negative recipient, peripheral blood was stained with Bw6 tetramer to detect Bw6-specific CAR Tregs. Dot plots show CD4+ CD8− FoxP3+ gated cells in peripheral blood at the indicated time points.(B and C) Detection of Bw6-specific CAR molecule on CD4+ CD8− FoxP3+ gated cells in peripheral blood (B) or bone marrow (C) at the indicated time points following adoptive transfer of Bw6-specific CAR Tregs into rapamycin and IL-2 preconditioned, antigen-negative animal.(D) Flow cytometry of CXCR4 expression from three separate Bw6-specific CAR Treg expansion products and unstimulated CD4s.