Schematic overview of the generation of rationalized CORSET8 transgenic mice.

I) Selection of the immunogenic epitope: here based on T cell epitope mapping results of Zhuang et al. (2021), which were confirmed by Erez et al. (2023) (Erez et al., 2023; Zhuang et al., 2021). II) Immunisation protocol to render Spike reactive T cells (for more information see Material and Methods section). III) Fluorescence-activated cell sorting (FACS) followed by single cell RNA and TCR sequencing. IV) Linking the single cell RNA and TCR sequencing data allows an integrated analysis to identify clonotypes and compare functional characteristics. V) Injection of the synthetic DNA into a fertilized oocyte followed by transfer into recipient females for gestation, resulting into the birth of transgenic founder offspring. VI) Dual evaluation of the different founders by phenotypical characterisation of the transgenic mouse and functional testing of the TCR Tg T cells. Parts of the figure adapted from 10xGenomics.

Clone identification using DALI assisted combined single cell and TCR analysis

A) Uniform manifold approximation and projection (UMAP) of splenic CD8 T cell single-cell RNA sequencing visualizing four different clusters based on single cell analysis. B) Projection of the three sequenced samples on the UMAP: CD8+ Tetramer positive cells of an immunized mouse (sample 1), CD8+ Tetramer negative cells of an immunized mice (sample 2) and total CD8 T cells from a naive mouse (sample 3). See Supplemental Figure 2 for detailed gating strategy of above-mentioned samples. C) Gene RNA expression in splenic CD8 T cells. Hallmark genes among the top differentially expressed genes are depicted. D) Overview of DALI pipelines using either -1-loading of the R Seurat object and immune profiling data directly into the interactive Shiny app or -2-generation of an extended R Seurat object containing immune receptor profiling data, which can be loaded into the interactive Shiny app -3-. E) UMAP of subsetted and reclustered Tetramer+ CD8 T cells showing three different clusters. F) RNA expression in Tetramer+ CD8 T cells of curated activation markers. G) UMAP of Tetramer+ CD8 T cells highlighting clonotype expansion. H) Clonotype frequency in the three different clusters of Tetramer+ CD8 T cells. I) Projection of clonotype1 on the UMAP of Tetramer+ CD8 T cells. J) TCRα and TCRβ sequence information of clonotype 1.

CORSET8 mice exhibit skewing to CD8 T cells and TCR Tg cells recognize target peptide ex vivo

A) Proportion of CD4 T and CD8 T cells among CD3+ TCRβ+ T cells in the spleen of CORSET8 mice and wildtype C57BL/6 littermates. B) Proportion of CD62L+ CD44-, CD62L+ CD44+ and CD62- CD44+ T cells among splenic CD4 T and CD8 T cells. C) Spike Tetramer staining on splenic CD8 T cells of CORSET8 mice or wildtype littermates. D) CTV proliferation profile of CORSET8 T cells in co-culture with BM-DCs in presence of increasing doses of Spike peptide. E) Visualization of CD4 and CD8 T cells in the thymus of CORSET8 mice and wildtype littermates. Data information: data are shown as means +/- SEM. After assessing normality by a Shapiro-Wilk normality test, parametric data were analysed with a t-test, whereas non-parametric data were analysed with a Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative of three independent experiments.

CORSET8 cells proliferate in vivo in response to Spike immunization or SARS-CoV-2 infection

A-B) Schematic set-up of the experiments. C) CTV dilution profile 4 days after intratracheal immunization with Spike/CpG or intramuscular immunization with Pfizer BNT162b2 vaccine. Quantification of the number of CORSET8 T cells and percentage of divided cells upon immunization with CpG control, Spike/CpG and Pfizer BNT162b2 vaccine. D) Mean fluorescence intensity (MFI) of several activation markers (CD44, CD69, IFN-γ, T-Bet) and percentage of Ki-67+ cells on both CORSET and endogenic CD45.2 T cells after i.t. and i.m. immunizations with Spike/CpG and Pfizer BNT162b2 vaccine, respectively. E) Schematic set-up of the experiment and gating on CD45.1+ CORSET8 cells in K18-hACE2wt/wt and K18-hACE2Tg/wt mice upon SARS-CoV-2 or mock infection. F) CTV dilution profile in SARS-CoV-2 infected K18-hACE2Tg/wt mice. G) Number of CORSET8 T cells in K18-hACE2wt/wt and K18-hACE2Tg/wt mice upon SARS-CoV-2 or mock infection. H) MFI of CD44 in CD45.1+ CORSET8 T cells and CD45.2+ T cells of recipient mice. I) MFI of Tetramer staining on CD45.1+ CORSET8 T cells and CD45.2+ endogenous Tetramer+ T cells. Data information: data are shown as means +/- SEM. In Fig. 4D, after assessing normality by a Shapiro-Wilk normality test, parametric data were analysed with a one-way ANOVA, whereas non-parametric data were analysed with a Kruskal-Wallis test. In Fig. 4H, after assessing normality by a Shapiro-Wilk normality test, non-parametric data were analysed with a Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative of two independent experiments.

Screening of CD8 T cell hybridoma clones and validation of first generation CORSET8 mice

A) MFI of IL-2 and IFNγ for individual CD8 T cell hybridoma clones. Based on the fold increase of IL-2 and IFNγ between medium control and Spike peptide pool stimulation, clone 47 was selected to proceed with. B) CD69 expression in hybridoma clone 47 in medium control and upon Spike peptide pool stimulation. C) BM-DC – CORSET8 T cell co-culture in presence of increasing dose of Spike peptide and Spike protein. D) Representative FlowJo plots gating on CD8 T cells in Rag2+/+, Rag2-/-, CORSET8 Rag2+/+ and CORSET8 Rag2-/- mice. Plotted cells are non-debris, single cells, viable and CD45+.

Gating strategy used to sort Spike Tetramer positive and Tetramer negative splenic CD8 T cells. Flow cytometry plots from a naive and a representative immunized mouse are shown. In total, three samples were subjected for single cell analysis; 1: Tetramer positive, CD44+ cells pooled from two immunized mice, 2: Tetramer negative cells pooled from two immunized mice, 3: Tetramer negative cells from one naïve mouse.