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

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, TGFβ, 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.