Supplementary Figure S6 from Overexpression of an Engineered SERPINB9 Enhances Allogeneic T-cell Persistence and Efficacy

Supplementary Figure S6. Overexpression of SB9(CAS) in ‘donor’ CD30.CAR with IgG1 spacer (CD30.Ig) EBVSTs increases resistance against allorejection by HLA-mismatched CD30-intact ‘recipient’ PBMCs. A, CD30.Ig expression on EBVSTs expressing different variants of SB9, measured by flow cytometry analysis of a representative donor. B, Pooled data of CD30.Ig expression (%) (B, left) and CD30.Ig expression (geometric mean fluorescence intensity, MFI) (B, right) on CD30.Ig EBVSTs, measured by flow cytometry analysis of n = 2 donors, where bars denote mean ± SD. C, SDS-PAGE analysis of SB9 monomer (∼43 kDa) and calnexin (∼90 kDa) expression. D, Densitometry analysis of SB9 monomer normalized to CD30.Ig EBVST control, using calnexin as a loading control. The bars denote mean ± SD. E, Schematic of in-vitro MLR setup for (F-I), where ‘donor’ CD30.Ig EBVSTs and ‘recipient’ HLA-mismatched PBMCs mixed in a 1:10 ratio and cell counts were monitored by flow cytometry over 12 days. The MLR was performed in the presence of IL-2 at 100 IU/mL, and the cultures were expanded when necessary. F, Representative flow plots showing total frequency of donor CD30.Ig EBVSTs on day 0 and day 12 of co-culture. G, The day 12 ‘donor’ cell death percentage, calculated by (donor cell count(monoculture)-donor cell count(co-culture))/(donor cell count (monoculture))*100%. Percentage of CD3+ ‘recipient’ T cells expressing CD30 (H) and CD3-CD56+ ‘recipient’ NK cells expressing CD30 (I). For G-I, each point represents a unique ‘donor’-‘recipient’ pair, and the bar chart represents the median (n = 4 per treatment group). P values were determined by one-way ANOVA, followed by Tukey post-test (G). *, P < 0.05; **, P < 0.01.