Panel A shows heatmaps of CD25 positive CD71 positive percent across soluble antibody, K562 CD16, K562 CD32, and K562 CD64 conditions for CAR and CAIleR constructs 5, 8, 10, and 11 at 0.5 micrograms per milliliter. K562 CD64 condition shows the highest activation reaching approximately 100 percent for CAIleR 8 and 10 when combined with their corresponding non-competitive antibody, while K562 CD16 shows the lowest antigen presenting cell signal and soluble antibodies minimal signal across all constructs. Panel B shows CAlleR 8 T cell flow cytometry plots of CD25 and CD71 expression when co-cultured with K562 CD32 and K562 CD64 and mAb 10 at different concentrations ranging from 0 to 1 microgram per milliliter, showing progressive increase in double positive populations with increasing concentration. Panel C shows sigmoidal curves of CD25 positive CD71 positive percent for K562 CD32 and K562 CD64, with EC50 values of 0.1027 and 0.0013 micrograms per milliliter respectively. Panel D shows flow cytometry plots of OX40 versus 41BB for polyclonal, CAR CD19, and CAlleR 8 Tregs with K562 CD16, CD32, and CD64, where CAIleR 8 shows 10.9 and 48.7 percent double positive with K562 CD32 and CD64, with significant differences marked. Panel E shows mCherry versus CD16, CD32, and CD64 plots confirming CAlleR 8 trogocytosis of Fc gamma receptors and expression of 12.4 and 19.0 percent for CD32 and CD64 respectively, with significant differences marked. Panels F and G show CFSE dilution histograms and suppression line graphs for K562 CD32 and K562 CD64 across CAlleR 8 Treg to Teff ratios from 1 to 1 to 1 to 32, where CAIleR 8 at 0.25 micrograms per milliliter reaches approximately 40 to 60 percent suppression at 1 to 1 ratio for both conditions, declining at higher dilutions.
FcγR-dependent antibody-mediated activation of CAlleRs. (A) Heatmaps showing mean expression of CD25 and CD71 activation markers on CAlleR T cells upon 48-h coculture with Bet v1 and anti-Bet v1 antibodies with or without K562 expressing three different FcγRs. (B) Representative plots of CD25 and CD71 expression on CAlleR 8 T cells upon 48-h coculture (n = 3) with different concentrations of mAb 10 and Bet v1 (0.5 μg/ml) in the presence of K562 expressing CD32 or CD64. (C) Dose–response curves with EC50 of CAlleR 8 T cell activation in the presence of K562 expressing CD32 or CD64, Bet v1 (0.5 μg/ml), and different concentrations of mAb 10 (n = 3). (D) Representative flow cytometry plots and cumulative data of 41BB and OX40 expression on CAlleR 8 Treg after 24-h coculture with K562 expressing different FcγRs, mAb 10 (1 μg/ml), and Bet v1 (0.5 μg/ml) (n = 3). (E) CAlleR 8–mediated trogocytosis of FcγR on Tregs after 24-h coculture with different K562, Bet v1 (0.5 μg/ml), and mAb 10 (1 μg/ml) (n = 3). (F and G) Suppressive effect of CAlleR 8 and polyclonal Tregs on CAlleR 8 Teff upon 96-h coculture with K562 expressing either (F) CD32 or (G) CD64 (K562:Teff ratio of 1:10), Bet v1 (0.5 μg/ml), and different concentrations of mAb 10. Plots show representative CFSE dilutions of CAlleR 8 Teff and cumulative data of the percentage of proliferation suppression. In A–E, coculture of K562 with T or Tregs was performed at a 1:5 ratio. Data represent the mean ± SEM from three to four independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test in D and E and by two-way ANOVA with Dunnett’s test to compare CAlleR 8 with polyclonal Tregs in F and G. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.