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. 2023 Sep 4;96:104785. doi: 10.1016/j.ebiom.2023.104785

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© 2023 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 3 — Specific killing of primary B target cells by autologous CD8+ T cells mediated by RoVER^(CD19). a) Graphics of the workflow for in vitro target cell killing assays using the RoVER technology (Created with BioRender.com). b)In vitro cell killing assays investigating RoVER^(CD19)-mediated killing of primary target CD19+ B cells using autologous CD8+ T cells obtained from YF-17D vaccinated study participants (n = 5). c)In vitro target cell killing of CD19+ B cells by autologous CD8+ T cells from healthy donor PBMCs using the FDA-approved BiTE, blinatumomab (n = 3). d) The dependence of RoVER^(CD19)-mediated target cell killing for YF epitope-specific CD8+ T cells was investigated in vitro target cell killing assays using CD8+ T cells obtained pre and post YF-17D vaccination (21 ± 3 days) (bar graph: n = 3, XY-graph: n = 7). To investigate potential differences in RoVER-mediated killing capacity using two bispecific RoVER^(CD19) containing different HLA class I-restricted YF epitopes together with autologous cells obtained from YF-17D vaccinated donors with matched HLA class I, we investigated in vitro target CD19+ B cell killing assays using a e) bispecific RoVER^(CD19_A2) engaging CD19+ B target cells and NS4B-HLA-A∗02:01-specific CD8+ T effector cells (n = 3), or f) bispecific RoVER^(CD19_B35) containing a NS2B-HLA-B∗35:01 domain (n = 2). g) HLA class I-specificity of RoVER^(CD19)-mediated killing was tested in vitro cell killing assays using RoVER^(CD19) containing a HLA class I domain matched to (HLA_pos) or different from (HLA_neg) the donor HLA class I (n = 3). h)In vitro cell killing assays investigating peptide- and RoVER^(CD19)-mediated killing of B cells using autologous CD8+ T cells obtained pre (n = 3) and post vaccination (21 ± 3 days) (n = 3, LLWNGPMAV; n = 4, SIINFEKL, −/+ RoVER). Data are the percentage of dead (Live/Dead stain) target cells (Celltrace+) out of total target cells shown as mean ± SEM for all donors (bar graph) or for individual donors (XY-graph) at the E:T or YF-E:T ratios indicated. p-values were calculated using Wilcoxon test. The dotted line is calculated based on the bars on the left side of each diagram and indicates the mean background target cell death.