Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Specificity of RLP-13 in dual-color co-cultures. (A) Setup of dual-color co-cultures: K562 cells transfected with covalently linked peptide:HLA-E expression constructs, presenting either cognate (Mtb44) or non-cognate (SP-2A) peptide, are stained with CellTrace Violet (CTV) or CFSE, respectively, and cultured for 18 hours with pre-expanded CD8 + T cells and different concentrations of RLP-13 at a ratio of 4:1:3 (Effector : Cognate target : Non-cognate target). Cells are then stained with lineage and activation markers and viability dye for analysis by flow cytometry. (B) Representative flow plot of viable single cell populations from co-culture wells without (left) or with (right) RLP-13. Indicated cognate and non-cognate gated population frequencies were used to calculate the relative viability of target cell populations. (C) Target cell viability of indicated cognate and non-cognate populations from co-cultures with various concentrations of either RLP-13 (red and pink lines) or irrelevant control scDb H2-mu (black and green lines). Viability was calculated by normalizing the frequency of the indicated target cell populations to that observed in co-culture wells without any scDb added. Assay was repeated five independent times across two HIV-negative donors. Shown measurements are averaged over eight technical replicates from one representative experiment. (D) Concentrations of the indicated effector molecules in the supernatants of co-cultures shown in (C) measured using the LegendPlex CD8/NK cell cytokine panel kit.

Article Snippet: For dual-color co-cultures, target cells were stained with CellTrace Violet or CFSE (dilution 1:20,000, FisherScientific, Cat. # C34557 and Cat # C34554) for 15 minutes in PBS at 37°C prior to plating.

Techniques: Transfection, Expressing, Construct, Staining, Cell Culture, Activation Assay, Flow Cytometry, Single Cell, Co-Culture Assay, Control