Mhc 1 m1 42

Thymocytes were stained for the expression of CD4, CD8α, TCRβ, CD69, and CCR7, and were sorted on FACS Aria II (BD Biosciences). Sorted cells were labeled with TotalSeq C hashtag oligos specific for CD45 (30-F11; BioLegend) and MHC-I (M1/42; BioLegend). Single-cell 5′ transcriptomic profiling was performed by using 10× Genomics system, and the data were analyzed by using RStudio software.

After animal sacrifice and perfusion with ice cold PBS, half of the brain of mice was collected and homogenized at defined times p.i. and leukocytes enriched on a Percoll gradient as previously described (Blanc et al., 2014 (link)). Cells were stained using the following antibodies: CD4 (GK1.5, Biolegend), CD3 (145–2 C11, Biolegend), IFN-γ (XMG1.2, Biolegend), FoxP3 (FJK-16s, eBioscience), IL-10 (JES5-16E3, Biolegend), CD8 (53-6.7, Biolegend), CD45 (30-F11, Biolegend), F4/80 (BM8, Biolegend), CD11b (M1/70, Biolegend), CD40 (HM40-3, Biolegend), CD86 (GL-1, Biolegend), MHCI (M1/42, Biolegend), and MHCII (M5/114.15.2, Biolegend). Virus-specific T cells were determined via flow cytometric analysis through either intracellular staining for IFN-γ or defined tetramers specific for immunodominant CD4 +and CD8+T cell-specific viral epitopes (Chen et al., 2014 (link); Stiles et al., 2006 (link)). Samples were analyzed using BD LSRFortessa and FACSDiva software and data was measured using FlowJo.