Figure 3.

Ag-scaffolds efficiently expand virus-specific T-cells and render a multifunctional T-cell product. (A) Number of viable cells at culture initiation (Unexp.) and following 2-week expansion with either dextran:pMHC:IL-2:IL:21 Ag-scaffold or free peptide-based expansion, adding specific peptide, IL-2 and IL-21 directly to the media, across 12 healthy donors in duplicate cultures. (B) Post-expansion frequency and (C) number of virus-specific T-cells from 15 parallel expansions in duplicate, covering 9 different virus-specific T-cell populations in PBMCs from 12 healthy donor buffycoats (BC), with either Ag-scaffold or free peptide-based expansion. (D) UMAP plot of HLA-A0101 CMV p50 VTE-specific T-cells expanded in duplicate from two healthy donors with either Ag-scaffold or free peptide. Specific T-cells were identified by tetramer-staining and stained with an antibody panel including markers of self-renewing (TCF-1, CD28, CD27) and terminally differentiated/exhausted (Tox, PD-1, GzmB) T-cells. The expression of these markers on Ag-scaffold (red) and free peptide (blue)-expanded T-cells were compared in MFI histograms. (E) Broadened phenotypic comparison between Ag-scaffold and free peptide-expanded T-cells (Log(MFI_Ag-Sc/MFI_FP) across four different virus-specific T-cell populations expanded in duplicate from four healthy donors. (F) Frequency of TCF-1⁺CD28⁺CD27⁺ and PD-1⁺GzmB⁺Tox⁺ T-cells out of antigen-specific T-cells expanded with either Ag-scaffold and free peptide. (G) Frequency of Triple+, Double+ and Single+ for IFN-γ, TNF-α and CD107a+ out of the total CD8+T-cell population on challenge with cognate antigen in expansions of HLA-A0201 CMVpp65 NLV-specific and EBV BRLF1 YVL-specific T-cells from eight healthy donors. (H) Single-cell phenotypic analysis and (I) dysfunctional score in a UMAP plot of unexpanded or free peptide-expanded and Ag-scaffold-expanded HLA-A0301 Influenza NP IRL-specific and HLA-B0702 CMV pp65 TPR-specific T cells from two healthy donor BCs, respectively. Duplicate-means were compared in a paired, non-parametric Wilcoxon test and p<0.05, p<0.01, p<0.0001 is indicated as *, ** and ****, respectively. Ag-scaffolds, antigen-presenting scaffold; BCs, buffycoats; CMV, cytomegalovirus; EBV, Epstein-Barr virus; GzmB, granzyme B; HLA, human leukocyte antigen; IL, interleukin; IFN, Interferron; MFI, mean fluorescence intensity; NLV, first three amino acids of peptide; PBMCs, peripheral blood mononuclear cells; pMHC, peptide-MHC; PD-1, Programmed cell death protein 1;TCF, transcription factor T-cell factor 1; TNF, tumor necrosis factor; Tox, Thymocyte selection-associated high mobility group box protein; UMAP, uniform manifold approximation and projection; VTE, first three amino acids of peptide