Extended Data Fig. 7: βII-spectrin expression in T cells.

a. Generation of GFP-ROSA^{βII-spectrin} mouse model (Rosa26^tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. b & c. Flow cytometry analysis of IFNγ and TNF expression in hHER2-CAR transfected T cells from GFP-ROSA^{βII-spectrin} mouse (T_{βII-spectrin}) after treated with PBS or Tat–Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. d. CAR-mediated B16-HER2 cell killing assay with T_{βII-spectrin} cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. e. Activated CD4⁺ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. f. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. g. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. h. Expression levels of βII-spectrin in human CD8 memory T cells (CD8⁺CD45RO⁺CD45RA^–CD56^–CD57^–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

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