Fig. 1

Neuroinvasive Lm infection induces accumulation of brain CD8⁺ T_RM cells. a Male C57BL/6J mice (n = 9) were infected with 2.0 × 10⁵ CFU Lm EGD and treated with antibiotics. Uninfected mice (n = 4) were injected i.p. with PBS then also received antibiotics. Mice were euthanized 29d p.i., and brain leukocytes were quantified by FACS. Symbols represent individual mice. Significance was calculated via two-tailed Student’s t test. b–d At 29d p.i., mice were/were not injected i.v. with CD45-PE/Dazzle 10 min prior to euthanasia as indicated. For this proof-of-concept experiment, some brains were harvested without perfusion prior to enzymatic digestion and FACS analysis of leukocytes. TheCD45^hiCD11b^neg-low population (box 1) was further gated on CD3⁺ to identify T lymphocytes. Discrimination between intravascular and extravascular cells was determined by staining with an isotype control. b Representative dot plot of a mouse that did not receive intravenous CD45, used as a negative control. c Representative dot plot of an unperfused mouse that received i.v. PE/Dazzle. CD3⁺ cells stained by PE/Dazzle were considered intravascular (gate 2), and CD3⁺ cells stained by PE/Dazzle were considered extravascular (gate 3). d Representative dot plot of a perfused mouse that received i.v. PE/Dazzle. After perfusion, 97.5 ± 0.7% of CD3⁺ cells were unstained, indicating they were extravascular (mean ± SD, n = 5). e Representative dot plots of extravascular CD3⁺ cells in a mouse that received i.v. PE/Dazzle. Extravascular CD3⁺ cells were gated on CD8⁺. These cells were predominately CD44⁺CD62L⁻ (f), and CD69⁺CX3CR1⁻ (g)