Identification, characterization, and expansion of innate lymphoid cells derived from canine peripheral blood...

Innate lymphoid cells (ILCs), a family of innate immune cells of lymphoid origin, serve as first responders to tissue damage and inflammation. They play a crucial role in defending mammals against tumor growth. ILCs can migrate between organs, move within tissues, and replenish from the blood during inflammation. Although much is known about ILCs in humans and mice, their properties in dogs are still largely unclear. To characterize canine ILCs, peripheral blood mononuclear cells (PBMCs) were isolated and cultured to expand ILCs. PBMCs were stimulated with recombinant human interleukin-2 (rhIL-2) and recombinant canine interleukin-7 (rcIL-7) for ILC1s expansion, rcIL-33 for ILC2s, or rh/rcIL-23 for ILC3s. Flow cytometry identified ILCs in PBMCs and cultures. We demonstrated that fresh PBMCs contained distinct populations of ILC1s, ILC2s, and ILC3s, characterized by the expression of cell surface markers (Lin^-, CD45⁺, CD127⁺) and the subset-specific transcription factors T-bet⁺ (ILC1s), GATA-3⁺ (ILC2s), and RORγt⁺ (ILC3s). Following the expansion on days 0, 7, 14, and 21, the purity of each ILC subset increased, peaking on day 14. The expanded ILCs upregulated subset-specific transcription factors, indicating differentiation and enrichment. These findings provide a reliable method to isolate, characterize, and expand canine ILCs from blood, useful for research on their roles in health, disease, and potential therapies. They also offer insights into human cancer research, as dogs develop cancers similarly to humans.