RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

Tzaban, S.; appasamy, p.; Zisman, E.; Klein, S.; Lewis, R.; Yu, H.; Khorgade, A.; Schwartz, M. A.; Sade-Feldman, M.; Eisenhaure, T.; Parnas, O.; Popovtzer, A.; Cohen, C.; Shifrut, E.; AlKhafaji, A. M.; Karni, R.; Eisenberg, G.; Hacohen, N.; Lotem, M.

doi:10.1101/2025.09.07.674706

bioRxiv · 10.1101/2025.09.07.674706

RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

Tzaban, S.·appasamy, p.·Zisman, E.·Klein, S.·Lewis, R.·Yu, H.·Khorgade, A.·Schwartz, M. A.·Sade-Feldman, M.·Eisenhaure, T.·Parnas, O.·Popovtzer, A.·Cohen, C.·Shifrut, E.·AlKhafaji, A. M.·Karni, R.·Eisenberg, G.·Hacohen, N.·Lotem, M.

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Abstract

Immune checkpoint blockade has transformed cancer therapy, yet many patients fail to respond, and few new targets have emerged beyond PD-1 and CTLA-4. Alternative splicing dramatically diversifies the T cell proteome, but the functional roles of most isoforms remain unknown. Here we constructed the first single-cell splicing atlas of human CD8 T cells, capturing dynamic isoform programs across activation and subset differentiation. This revealed distinct splicing footprints that refine conventional transcriptomic states and highlight receptor families with isoform-level regulation. To functionally interrogate these candidates, we developed SpliceSeek, a CRISPR-based pooled screening platform that perturbs splice sites to redirect isoform usage. Using SpliceSeek, we uncovered isoform-specific immune checkpoints whose perturbation enhanced effector function and tumor control, including the LRRN3-203 isoform, which augmented cytokine secretion and antitumor immunity in mice models. Together, our results establish alternative splicing as a targetable layer of immune regulation and demonstrate the potential of isoform-focused screening to expand the landscape of cancer immunotherapy.

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Tzaban, S., appasamy, p., Zisman, E., Klein, S., Lewis, R., Yu, H., Khorgade, A., Schwartz, M. A., Sade-Feldman, M., Eisenhaure, T., Parnas, O., Popovtzer, A., Cohen, C., Shifrut, E., AlKhafaji, A. M., Karni, R., Eisenberg, G., Hacohen, N., Lotem, M.. 2025-09-11. RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy. https://doi.org/10.1101/2025.09.07.674706

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