*Purpose: Allogeneic invariant Natural Killer T cells (allo-iNKT) are attractive for transplant as they prevent graft rejection and maintain anti-tumor/pathogen effects but do not induce GVHD. However, their safety and schedules for clinical use are unknown. As dogs have proven valuable in developing protocols for human allotransplant, we investigated the safety, feasibility and effects of adoptive transfer of canine allo-iNKT to inform their use in human patients.

*Methods: We comparatively evaluated the phenotype and function of ex-vivo expanded canine and human iNKTs by flow cytometric immunoassays and single-cell RNA-sequencing (scRNA-seq). 4×10⁸ sex-mismatched canine allo-iNKTs were transferred into a healthy male recipient dog to enable donor iNKT tracking by qPCR of the female specific Xist gene. Safety was determined by clinical, hematological and biochemical analyses. Effects on host immune populations were monitored by flow cytometry.

*Results: Results: In vitro assays revealed that canine iNKTs have similar phenotype and function to human iNKTs. For adoptive transfer, we selected a donor whose iNKTs had the greatest cytotoxic potential and expandability based on flow cytometry (mostly CD8⁺), in vitro proliferative response (nearly 10⁶ fold expansion in 3.5 weeks) and scRNA-seq profiling (predominant killer and Th1 features over Th2). 4×10⁸ allo-iNKTs were infused into an unrelated 30kg dog after non-lymphodepleting preconditioning (cyclophosphamide 250mg/m2). Cell therapy was well tolerated with no evidence of cytokine release syndrome. Within 24h, peripheral blood iNKTs had a fivefold increase. Neutrophils and monocytes decreased over the next 10 days whilst lymphocytes steadily increased. Lymphoblast-like cells appeared 7 days after allo-iNKT therapy. Blood counts and flow cytometry confirmed expansion of NK, CD8⁺T, Treg and MDSC. By contrast, only mild iNKT and Treg increases occurred in another dog with treated the iNKT agonist α-Galactosylceramide instead of allo-iNKTs.

*Conclusions: For the first time, we have shown that off-the-shelf canine iNKT cells can be generated to scale, cryopreserved and safely transferred. Remarkably, allo-iNKTs induced expansion of autologous regulatory and killer cells in vivo. These findings pave the way to evaluate allo-iNKT protocols and mono/combination therapies in canine patients to inform human clinical use.

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