As the first analysis step, macaque leukocytes with similar phenotypes were grouped together into cell clusters using the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm. Phenotypically similar cell clusters are connected in a minimum spanning tree.

The SPADE tree can be colored based on the expression of 15 markers used for cell population annotations. An annotated SPADE tree is also provided.

SPADE clusters were annotated with respect to the expression of 15 markers and as follows: neutrophils (CD66high CD125-), neutrophils (CD66mid CD125+), basophils (CD66-CD123+HLA-DR-), monocytes (CD14+HLA-DR+), cDCs (CD14-HLA-DR+CD11c+CD16+), inflammatory cDCs/non-classical monocytes (CD14+HLA-DR+CD11c+CD16+), pDCs (CD123+HLA-DR+), CD141+ CADM1- APCs (CD3-CD8-CD14-CD20-CD11c-CD16-CD123-CD141+CADM1-HLA-DR+), CADM1+ APCs (CD3-CD8-CD14-CD20-CD11c-CD16-CD123-CADM1+HLA-DR+), B cells (CD20+HLA-DR+), T cells (CD3+), and NK cells (CD3-CD8+).

Granulocyte and monocyte and DC clusters were ‘isolated’ from this first clustering analysis to be further studied. We defined granulocytes as neutrophils and basophils and monocytes-DCs as monocytes, cDCs, pDCs, inflammatory cDCs/non-classical monocytes CD141+CADM1- APCs and CADM1+ APCs.