Engineered iMSCs delivering wild-type IL-2 achieve dose-sparing tumor control via CD25-dependent CD8⁺ T-cell...

Interleukin-2 (IL-2), the first FDA-approved cytokine-based immunotherapy, can induce durable antitumor responses but its broader use is curtailed by life-threatening, dose-dependent toxicities. This narrow therapeutic window has limited clinical translation for decades. Genetically engineered mesenchymal stromal cells (MSCs) have emerged as attractive IL-2 delivery vehicles due to their low immunogenicity and intrinsic tumor-homing capacity.

Human induced pluripotent stem cells (iPSCs) were edited by CRISPR/Cas9 to knock in wild-type IL-2 (wtIL-2) or a receptor-biased variant (IL-2v) into the B2M or B3 safe-harbor loci, generating cytokine-secreting iPSC-derived MSCs (iMSCs). Antitumor activity was evaluated in co-cultures of iMSCs, tumor cells, and peripheral blood mononuclear cells (PBMCs) by flow cytometry. In vivo, systemically administered iMSCs were tracked by live imaging in mice bearing subcutaneous tumors to assess biodistribution and tumor targeting. Tumor control was monitored by longitudinal tumor growth and end-point volume; toxicity was evaluated by lung wet weight and blood biochemistry. Immune cell states within the tumor microenvironment were characterized by flow cytometry and RNA sequencing. Data (mean ± s.e.m.) were analyzed in GraphPad Prism using t-tests, ANOVA, or Kruskal-Wallis tests, as appropriate (n = biological replicates; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Surprisingly, we found that under low-dose conditions, only iMSCs with IL-2 overexpression-but not unmodified iMSCs or free IL-2 alone-induced robust upregulation of CD25 on CD8⁺ T cells. This effect likely increases T-cell sensitivity to wtIL-2 and may contribute to the improved tumor control achieved at reduced IL-2 doses. Systemic administration of wtIL-2-iMSCs preferentially activated intratumoral T cells and achieved tumor growth inhibition comparable to high-dose rhIL-2 in our model, while attenuating systemic toxicity. Transcriptomic analysis indicated that wtIL-2-iMSCs enhanced effector programs in tumor-infiltrating CD8⁺ T cells more effectively than rhIL-2 under the tested conditions. Notably, targeted integration of wtIL-2 at a novel locus within the B2M region (B3-wtIL-2-iMSCs) led to a higher transgene expression and further augmented antitumor activity in vitro and in vivo with no clear increase of systemic toxicity. Moreover, B3-wtIL-2-iMSCs exhibited synergistic antitumor effects when combined with anti-PD-1 blockade, thereby strengthening overall tumor control.

These findings point to a mechanism by which iMSC-delivered IL-2 reshapes the tumor microenvironment through CD25-dependent CD8⁺ T-cell activation. Furthermore, the results support the concept that this platform enables a more dose-efficient cytokine delivery strategy with an improved safety profile, thereby facilitating the clinical translation of off-the-shelf allogeneic iMSC-based gene therapies.