Localization and kinetics of HVEM expressing cells in the spleen of VACV infected mice.

(A–D) Groups of C57BL/6 wild-type (WT) mice were infected i.p. with VACV-WR (3×10⁴ PFU/mouse). Uninfected (naïve) mice were used as controls. (A) Frozen sections of naïve (day 0), day 4, day 6 and day 8 VACV infected mice were stained with rat anti-mouse CD3-PE, HVEM-APC and CD169-FITC antibodies. The images were captured by 20× objective using EVOS fl inverted microscope. The micrographs arranged vertically in 1^st, 2^nd, and 3^rd column (left to right), showing localization of CD3 (red), HVEM (purple) and CD3+HVEM expression in the splenic white pulp respectively. CD169 (green) was used to identify splenic marginal zones. (B) B cell follicles (f) identified by B220 (green channel), perilymphatic sheath (PALS) identified by CD3 (red channel) and co-localization of HVEM and CD3 antibodies in PALS region of white pulp. On days 4, 6, 15, 64 (C), and day 120 (D) postinfection splenocytes were harvested and stained for CD8, CD44, VACV-specific tetramers (B8R, A8R, or B2R), and HVEM. Representative plots of HVEM staining on total CD8 T cells and tetramer (B8R, A8R, and B2R) positive cells after gating on CD8 cells. The numbers in each plot indicate the percentage of total CD8 T cells (CD44_low and CD44_high) or tetramer-positive cells that stained for HVEM.