Fig 2: Design and generation of PD-1CAR Tregs from a PD-1KO mouse CD4+ cell line and primary CD4+ cells(A) Schematic illustrating the 3-in-One PD-1CAR Treg design, which enables direct engagement, activation by, and immunosuppression of PD-1+ eTconvs, in contrast to conventional IndiCAR Tregs that require antigen recognition and activation before migrating to the vicinity of effector cells to exert their suppressive function.(B) Schematic of the PD-1CAR construct, highlighting key design features. Illustrations of (A) and (B) were created using BioRender.com.(C) Representative flow cytometry histograms shows scFv expression in PD-1CAR Tregs derived from PD-1KO 2D2 cells, compared to parent cells.(D) Representative flow cytometry dot plots illustrates the co-expression of GFP and scFv in PD-1CAR Tregs derived from PD-1KO 2D2 cells, compared to parent cells.(E) Representative flow cytometry histograms show FoxP3 expression in PD-1CAR Tregs derived from PD-1KO 2D2 cells, compared to parent cells.(F) Bar graphs summarizing the MFI of scFv on PD-1CAR Tregs derived from primary CD4+ cells of PD-1KO mice, compared to parent cells.(G) Quantification of FoxP3 expression, presented as MFI, of PD-1CAR Tregs derived from primary CD4+ cells from PD-1KO mice, compared to parent cells.(H) Measurement of CD25 expression, presented as MFI, in PD-1CAR Tregs derived from primary CD4+ cells from PD-1KO mice, compared to parent cells. Data of (F–H) are mean ± SEM, n = 3–6. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by Student’s two-tailed t test.

Fig 3: PD-1CAR Tregs Bind PD-1 for Engagement(A) Representative flow cytometry dot plots show PD-1 binding on 2D2 cell line-derived PD-1CAR Tregs, but not on parent 2D2 PD-1KO cells.(B) Dose-dependent binding of PD-1, represented as MFI, on 2D2 cell line-derived PD-1CAR Tregs, with no detectable binding on parent 2D2 PD-1KO cells.(C) Representative flow cytometry dot plots show double-positive (CFSE+/eFluor 670+) cell conjugates, indicating the binding of CFSE-labeled 2D2 cell line-derived PD-1CAR Tregs with eFluor 670-labeled PD-1+ or PD-1− EL4 cells.(D) Bar graph quantifying the percentage of CFSE+/eFluor 670+ double-positive cell conjugates formed between PD-1+ EL4 cells co-cultured with 2D2 cell line-derived PD-1CAR Tregs at a ratio of 1:1, compared to conjugates formed with PD-1− EL4 cells. Data of (B) and (D) are mean ± SEM, n = 4; ∗∗∗p < 0.001, ∗∗∗∗p < 0.000, as determined by Student’s two-tailed t test.

Fig 4: PD-1CAR Tregs inhibit primary CD4+ T cell proliferation and preferentially reduce the proportion of PD-1+ target cells(A and B) Representative flow cytometry histograms show the proliferation of mouse CD4+ T cells (pre-activated overnight with anti-CD3/CD28 beads at a 1:8 bead-to-cell ratio, followed by bead removal) co-cultured for 4 days with either primary PD-1CAR Tregs (A) or parent cells (B) at varying ratios.(C) Quantification of proliferation inhibition based on the division index (DI) calculated from data shown in (A) and (B).(D–F) Representative flow cytometry dot plots show PD-1 expression on PD-1+ target cells after four days of culture: (D) target cells alone, (E) target cells co-cultured with primary PD-1CAR Tregs, and (F) target cells co-cultured with control polyclonally induced Tregs. PD-1+ target cells of (D–F) were generated by pre-activating primary CD4+ T cells isolated from 2D2 transgenic mice with 3 μg/mL plate-bound anti-CD3/CD28 for three days and then labeled with CellTrace Far Red before the co-culture. Data of (C) are mean ± SEM, n = 3; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by Student’s two-tailed t test.