Journal: bioRxiv

Article Title: T cell engagers convert acute tumor killing into endogenous vaccination

doi: 10.64898/2026.10.06.756695

Figure Lengend Snippet: A . Pseudotime trajectory analysis (Monocle 3) overlaid on the UMAP from , suggesting a continuous differentiation path from pre-existing naïve and/or memory populations, through the progenitor-like state, to cytotoxic effectors. B-E: Murine adoptive transfer reveals that T CM display the highest potential for expansion and differentiation into CTL TCE . B . Experimental design: T Naïve (CD44⁻ CD62L⁺), T CM (CD44⁺ CD62L⁺) and T EM (CD44⁺ CD62L⁻) CD8⁺ T cells are sorted from LckGFP hCD3ε chimeras and transferred into hCD20⁺ tumor-bearing C57BL/6J recipients (non-chimeric) one day before TCE or isotype control administration. GFP⁺ transferred cells are analyzed at day 6 post-treatment. Each dot represents one mouse; data pooled from 2 independent experiments. P values calculated by one-way ANOVA after adjustment using Tukey’s multiple comparisons test. C-D : Characterization of recovered GFP + T cells at D6 from TCE or isotype control . From top to bottom: evolution of transferred T Naïve , T CM, and T EM subsets upon TCE administration. C. Representative flow cytometric gating of recovered GFP + T cell subsets and quantification among total T cells, for each transferred subset. D. Representative gating of phenotype by CD44 and CD62L expression among recovered GFP + CD8 + T cells, and expression of cytotoxic molecules allowing the quantification of CTL TCE (GFP⁺ CD8⁺ perforin + granzyme B⁺). FACSplots from isotype-treated mice are uncolored; TCE-treated FACSplots are colored according to the sorted subset transfer (gray: T Naïve ; blue: T CM ; orange: T EM ). E . Quantification of CTL TCE among total T cells depending on subset transfer, in the spleen (left) and the blood (right). F-H: The superiority of memory subsets over naïve cells for CTL TCE generation is conserved in human co-culture experiments. F . Experimental design for human validation: T Naïve (CCR7⁺ CD45RA⁺), T CM (CCR7⁺ CD45RA⁻) and T EM (CCR7⁻) CD8⁺ T cells sorted from healthy donor PBMCs are co-cultured with hCD20⁺ B cell tumor in the presence of TCE or isotype control. Analysis is performed after 3 days of co-culture. Two different donors from 2 independent experiments are pooled (Donor 1, circles; Donor 2, diamond). P values by one-way ANOVA with adjustment by Tukey’s multiple comparison test. G . Left: Representative histograms of granzyme B expression in CD8⁺ T cells upon treatment with isotype (empty plot) or TCE (full plot), according to the type of subset in culture. Right: Quantification of the gMFI of granzyme B expression in CD8⁺ T cells upon TCE exposure, normalized to the mean gMFI in the isotype control group. H . Killing index per subset upon TCE exposure, normalized to the mean killing index in the isotype control group.

Article Snippet: Six to eight-week-old male C57BL/6J mice were obtained from Envigo.

Techniques: Analysis, Adoptive Transfer Assay, Control, Expressing, Co-culture Assay, Biomarker Discovery, Cell Culture, Comparison, Cell Differentiation, Transmission Electron Microscopy, Co-Culture