Fig. 1. Unbiased clustering identifies upregulation of Ly6C on the surface of CD8⁺ Tn cells following WNV infection.

C57BL/6 mice (N = 9) were infected with 1000 PFU WNV virus by footpad injection. WNV infected and uninfected control mice (N = 9) were bled retro-orbitally at days 7 post-infection and stained with a 15 marker flow cytometry panel. FCS files were uploaded in Cytobank, cloud-based clustering software, CD3⁺ live cells were gated prior to clustering. Flowsom visualization algorithm identified the standard subsets of CD8⁺T cells effector/effector memory ((EM) CD44^hiCD62L⁻), central memory ((CM) CD44^hiCD62L⁺CD49d⁺), virtual memory ((VM) CD44^hiCD62L⁺CD49d⁻) and naïve ((N) CD44^loCD62L⁺CD49d⁻). Tn was divided into two subsets based on Ly6C expression, Ly6C⁺ subset (indicated by red arrow) was enlarged at d7 of WNV infection. Data is from one experiment (with N = 9 mice per group).