Figure S2.
Panel A shows a schematic of K562 cell line generation, where K562 CD32 undergoes CRISPR Cas9 mediated CD32 knockout to generate K562 CD16, followed by lentiviral transduction of an EF1 alpha CD64 construct to generate K562 CD64. Panel B shows flow cytometry plots of SSC-A versus CD16-PE, CD32-APC, and CD64-APC for K562 CD16, CD32, and CD64 cell lines, confirming specific expression with CD16 at 97.1 percent, CD32 at 98.0 percent, and CD64 at 91.3 percent. Expression of CD32 and CD64 remains below 0.38 percent across cell lines that do not express such markers. Panel C shows CFSE dilution histograms for K562 CD32 and K562 CD64 across mAb 10 concentrations of 0, 0.05, 0.1, and 0.25 micrograms per milliliter, where increasing concentrations show progressive CFSE peak shifts indicating greater proliferation suppression in K562 CD64 compared to K562 CD32.
Generation and validation of K562 cell lines expressing different FcγRs. (A) Transgenic K562 cell line generation. CD32 KO K562 cell line and generation of CD64 transgenic CD32 KO K562. (B) Flow cytometry plots showing CD16, CD32, and CD64 expression on the three cell lines. (C) Representative plots of human CAlleR 8 T cell proliferation after 96 h of coculture with K562 expressing CD32 or CD64 (K562:T cell ratio of 1:10) in the presence of Bet v1 (0.5 μg/ml) and different concentrations of mAb 10.