Fig 2: Flow cytometry gating strategy for identifying canine ILC subsets in PBMCs. The plots illustrate the sequential gating strategy used to identify canine ILCs. Cells were first gated on FSC/SSC characteristics to identify the lymphocyte population, followed by live-cell gating and doublet exclusion. CD45+ leukocytes were gated, and lineage-positive cells (CD3, CD5, CD21, and CD11b) were excluded. The remaining Lin−CD127+ population (Q1) was defined as total ILCs and used to identify canine ILC1, ILC2, and ILC3 subsets. Representative flow cytometric dot plots from the in vitro expansion of ILC1s are shown.

Fig 3: Distribution of ILC subsets after in vitro expansion. (A) Percentage of ILC subsets within Lin−CD45+CD127+ cells from 9 healthy dogs following 14 days of cytokine-supported expansion culture. Data represent technical replicates for each donor (18 total measurements). (B) Representative flow cytometric contour plots of expanded canine ILC1, ILC2, and ILC3 populations. Cells were gated on CD45+ lymphocytes and lineage-negative (Lin−; CD3−, CD5−, CD21−, CD11b−) populations. Within the Lin−CD45+ compartment, CD127+ cells were further analyzed for transcription factor expression to define ILC subsets: T-bet+ (ILC1), GATA3+ (ILC2), and RORγt+ (ILC3). Contour plots show the distributions of these subsets, highlighting clear separation by transcription factor expression (FMO, gray contours; samples, red contours) following in vitro expansion. Representative plots are shown from a single donor, with similar patterns observed across independent samples. Percentages of ILCs on the plots are shown as mean ± SD.

Fig 4: Number of circulating innate lymphoid cell (ILC) subsets per milliliter of peripheral blood in individual dogs. (A) Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from nine dogs. ILC subsets were identified as Lin− (CD3−CD5−CD21−CD11b−) CD45+ cells within the CD127+ population, expressing subset-specific transcription factors (T−bet+ in ILC1s, GATA−3+ in ILC2s, and RORγt+ in ILC3s) using flow cytometry. Data are expressed as cells per mL of whole blood. (B) Flow cytometry was used to determine the percentages of individual subsets (ILC1, ILC2, and ILC3) within canine PBMCs. Each dot represents an individual dog (n = 9). The Y-axis is shown on a linear scale.