Fig. 5

CXCR3 ligands are expressed at sites of early immune activation. a Schematic illustrating CD8⁺ T cell isolation, CXCR3 ligand (CXCR3L) stimulation, and longitudinal sampling for flow cytometric analysis. b Representative gating of OT-I T cells, NKG2D⁺ endogenous memory CD8⁺ T cells (blue), and NKG2D^– endogenous naive CD8⁺ T cells (gray). c Representative histograms depicting surface CXCR3 flow staining in OT-I T cells (black border), endogenous memory CD8⁺ T cells (blue border), and endogenous naive CD8⁺ T cells (gray border) after 30 min of mock (dark gray histogram) or CXCL9 plus CXCL10 (rCXCR3L) stimulation (10 ng/mL, light green; 50 ng/mL, medium green; 100 ng/mL, dark green per chemokine). d Frequencies of OT-I T cells (left), endogenous memory CD8⁺ T cells (center), and endogenous naive CD8⁺ T cells (right) expressing surface CXCR3 prior to rCXCR3L stimulation (0 min), after rCXCR3L stimulation (30 min), and 30 min after rCXCR3L removal (60 min). Symbols and line connectors are color-coded by rCXCR3L concentration as in c. e, f Representative IF image of spleen from animal 24 h post WT LM immunization. The image is composed of three 8 µm serial sections stained for CD11b (green) and CD169⁺ MMM (blue); OT-I T cells (red) and WT LM (white); and CXCL9 (magenta) and CXCL10 (cyan). c is a representative plot from one of two technical replicates. In d each symbol represents the mean (n = 2 at each timepoint) ± SD with lines connecting means from two technical replicates. e, f is representative of n = 5 animals from two technical replicates. Image orientation was modified in Adobe Photoshop to permit overlaying of serially sectioned slides. Contrast was equally increased amongst all individual images; after, punctate staining in LM, CXCL9, and CXCL10 images was outlined using ImageJ command “find edges” to enhance visibility prior to merging. See also Supplementary Fig. 7. Source data are provided as a Source Data file