人 CD8-iTreg 通过释放 Granzyme-K (+) 超分子攻击颗粒成为强效 GVHD 抑制细胞和杀肿瘤效应细胞

UNLABELLED: 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 , 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 SUMMARY: CD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4 + SMAPs containing granzyme K.