Fig 2: CD5-depleted and CD5dim cells retain gene expression related to broad innate immune cell function when compared to NKp46+ cells in steady-state. DGE was performed between canine cells isolated using CD5-depletion via magnetic separation, cells isolated via FACS for CD5dim and cells isolated via FACS for NKp46+ in the steady-state (at rest). The purpose of this experiment was to compare the gene expression between three most common starting populations described most frequently in the canine NK cell literature. (A) Individual normalized gene counts of canonical NK markers by treatment group. Within these classically NK-associated genes, CD5-depleted and CD5dim expression is similar and much lower than expression in NKp46+ cells, with the exception of PRMD1/BLIMP1 and ZEB2 which is higher in the CD5-depleted and CD5dim populations. (B) A heatmap of the top 30 most differentially expressed genes demonstrates a subset of genes induced in CD5dim cells, compared to the other two populations, none of which are classically associated with NK cell function. CXCL8 is the gene for interleukin-8 and most commonly secreted by macrophages. The G0S2 gene governs the G0/G1 switch and governs cell cycle progression in blood mononuclear cells. FTH1 encodes ferritin heavy chain associated with iron binding. (C) Principal component analysis demonstrates the variance between CD5dim and NKp46+ cells drives PC1 (86%), whereas the difference between CD5-depleted and CD5dim cells drives the variance for PC2 (7%), which is much smaller. (D) Gene-ontology (GO) analysis between the cell populations is depicted as UpSet plots. UpSet plots the intersections of a set as a matrix. In this case, each set on the left represents a gene pathway. The quantity in the bar graph represents the normalized number of counts for that gene pathway. When genes contribute to multiple gene pathways (for example, cell cycle and oocyte meiosis) a bar connects the dots in the matrix. Thus, performing GO analysis the cell pathways on the left of each graph denote the pathways induced in a specific population when compared to a second. Gene overlap between pathways depicted by connected dots. In this case, CD5-depleted and CD5dim cells share induced pathways related to broad innate functions, to include phagosome, lysosome and endocytosis. In contrast, NKp46+ cells appear to have induced pathways involved in T cell function when compared to CD5-depleted and CD5dim cells. None of the populations appear to possess induced pathways related to NK cell function when compared to the others. It is important to note this does not denote the absence of NK cell function in the populations, but rather the absence of significant differences in any NK cell functions between the three populations.

Fig 3: Stages of NK cell activation develop in a conserved trajectory. Diffusion mapping of cell populations illustrates a time-dependent diffusion process where the Euclidian distance between points approximates the diffusion distance. Populations used for cell sorting (resting CD5dim, resting CD3-NKp46+, activated CD3-NKp46+ and unsorted activated) are consistent with prior experimental conditions. (A) Here, we see an organized, uniform and quantifiable diffusion trajectory of activated CD3-NKp46+ and unsorted dog NK cells arising from resting CD5dim cell populations, depicting heterogeneity in the biologic response to co-culture stimulation. Clusters along the trajectory have been divided by natural break points. (B) Visualization of gene expression changes between trajectories on heatmap. Note each cluster has a unique transcriptomic profile. (C) The top 10 differentially expressed genes for each cluster were collected. Genes that were noted to be differentially expressed between more than 1 cluster (suggesting stepwise changes throughout activation) were identified. Visualization of these genes are shown as ridge plots, which demonstrate changes in gene expression of these driving genes over time. Six genes, including canonical NK genes, had significant DGE between recurrent clusters: CCL5, GZMA, IL2RB, ZEB2, KLRB1, and TYROBP (Dap12).

Fig 4: Single-cell analysis of resting CD5dim and CD3-NKp46+ canine cells confirms greater heterogeneity in CD5dim compared to the NKp46+ population. DGE following single-cell RNA sequencing of FACS-isolated resting CD5dim and CD3-NKp46+ cells from healthy beagle PBMCs. ScRNASeq was performed using the plate-based 3’ UPX Transcriptome kit with 24 cells/group given the comparative rarity of the CD3-NKp46+ group in peripheral blood. (A–C) PC analysis demonstrates overlap of the two populations based on their transcriptional signatures, indicating that the two populations are not unique. Variance in PCA is driven by outliers from the CD5dim population, indicating that this population is more heterogeneous and likely includes either additional subsets of NK cells or non-NK cells. Given the low percentage of variance seen in PC1, PC2 and PC3, p-values of the variance for each principal component were calculated and depicted in the associated Jackstraw plot (C). The p-value for each eigenvalue does not reach significance, and thus the populations are quantitatively transcriptionally similar. (D) Panel of genes differentially expressed between CD5dim and CD3-NKp46+ cells with the lowest p-values in the steady-state (resting). Note the genes in the left and middle columns pertain to the NKC gene complex and Granzyme A and B, and expression is similar, save for KLRK1/NKG2D. The non-NKC genes in the right column are driving the variance for outlying CD5dim cells. (E) Heatmap depicting qualitative gene expression changes between the two populations, for which no consistent pattern of variation is seen between top differentially expressed genes. (F) Heatmap depicting genes driving the variance for PC1 and PC2, representing how the gene expression of the outlying CD5dim cells differ from the main cluster. Outlying cells have decreased expression of cell cycle genes MERTK, CDK12, PIK3AP1, among others.

Fig 5: Canine CD5-depleted and sorted CD3-NKp46+ cell populations converge on identical gene expression in the activated state. Differential gene expression analysis of resting (Day 0) CD5-depleted and FACS-isolated CD3-NKp46+ versus activated (Day 14) NKp46+ and expanded CD5-depleted cells as candidate canine NK cell populations. (A) Individual gene counts of canonical NK markers by treatment group: CD5-depleted resting, CD3-NKp46+ resting, CD3-NKp46+ activated and unsorted expanded CD5-depleted activated cells. Sorting on CD5-depleted in the activated state is not possible as expression is lost after 14-days of feeder line co-culture. Resting CD3-NKp46+ cells have higher expression of canonical NK-associated genes than CD5-depleted cellsand therefore may represent a subset of circulating NK cells. (B) Flow cytometry results for CD5dim versus NKp46 staining of the starting population from healthy beagle PBMCs, demonstrating overlapping of the two phenotypes. (C, D, E) MA (ratio intensity) plot, mean of normalized counts by log fold change, compares differentially expressed genes (in red) between treatment conditions. In total, Figure 2B contains 2888 induced genes and 2109 repressed genes, Figure 2C contains 1893 induced genes and 1896 repressed genes, and Figure 2D contains just 1 induced gene and 7 repressed genes which are differentially expressed between the cell populations. Thectivated CD3-NKp46+ cells compared to activated unsorted cells in Figure 2D have very few differentially expressed genes and thus nearly identical transcriptional profiles. (F) Principal component analysis reveals resting CD5-depleted cells drive variance for PC1, while resting CD3-NKp46+ cells drive variance for PC2 (F). Almost no variance between activated CD3-(E). (G) The similarity matrix demonstrates Jaccard index (J(A,B) = |AnB| / |AUB|) approaching 1 (identical) between activated CD3-NKp46+ and CD5-depleted expanded cells based on pre-abundance count hash sketches. This signifies similarity based on fastq sequences prior to alignment to a transcriptome or normalization of counts. (Key: CD5dim_D0 = resting CD5dim cells, NKP46_D0 = resting NKp46+ cells, NKp46_D14 = coculture activated NKp46+ cells, US_D14 = unsorted coculture activated cells expanded from a CD5dim resting population.) (H) Dotplots of gene ontology analysis results (using KEGG) comparing gene pathways induced in resting CD5-depleted cells, resting CD3-NKp46+ cells and activated CD3-NKp46+ cells. Note that pathways induced in CD5-depleted cells include phagocytosis, endocytosis and lysosome functions which have been broadly implicated in innate immune cell pathways including NK cell trogocytosis. Also note resting CD3-NKp46+ cells express the NK cell-mediated cytotoxicity pathway, but also pathways associated with T-cell development. As before, activated CD3-NKp46+ cells express pathways associated with pathways associated with both high replication and cellular dysfunction.