Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles

Larson, J. H.; Compeer, E. B.; Dougherty, P. R.; Smith, K.; Zaiken, M. C.; Margaritaki, O.; Kopp, B.; Harkiolaki, M.; Jin, S.; Chen, L.; Valvo, S.; Staton, C.; Capitani, N.; Cassioli, C.; Payne, N. C.; Bolivar Wagers, S.; Hani, S.; Houle, B.; Peng, Y.; Baldari, C. T.; Kean, L. S.; Cantor, H.; Dranoff, G.; McDonald-Hyman, C.; Hippen, K. H.; Dustin, M. L.; Blazar, B. R.

doi:10.64898/2026.06.18.731665

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bioRxiv · 10.64898/2026.06.18.731665

Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles

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Abstract

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

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Larson, J. H., Compeer, E. B., Dougherty, P. R., Smith, K., Zaiken, M. C., Margaritaki, O., Kopp, B., Harkiolaki, M., Jin, S., Chen, L., Valvo, S., Staton, C., Capitani, N., Cassioli, C., Payne, N. C., Bolivar Wagers, S., Hani, S., Houle, B., Peng, Y., Baldari, C. T., Kean, L. S., Cantor, H., Dranoff, G., McDonald-Hyman, C., Hippen, K. H., Dustin, M. L., Blazar, B. R.. 2026-06-23. Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles. https://doi.org/10.64898/2026.06.18.731665

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