Supplementary Figure 2: Naive CD5^hi CD8⁺ T cells exhibit rapid production of the chemokine XCL1.

Splenocytes from T-bet-GFP reporter mice were left untreated or stimulated with PMA/ionomycin in the presence of brefeldin A. Cells were then stained for CD8, CD44 and CD5, and then fixed, permeabilized and stained intracellularly for XCL1 and also CXCR3 (since detectable surface expression of CXCR3 was reduced by stimulation). T-bet expression was determined using the T-bet-ZsGreen (enhanced GFP) reporter (note – some quenching of the ZsGreen reporter is apparent in stimulated cells). In other experiments (including those shown in Fig. 1a,b), T-bet was detected by intracellular staining, with similar results. (a) Expression of CXCR3 and T-bet (T-bet-ZsGreen reporter) was determined in unstimulated cells, while staining for XCL1 was assessed after PMA/Ionomycin stimulation. The cells were counterstained for CD5 and the data gated on CD44^lo CD8+ T cells. (b,c) Co-expression of XCL-1, CXCR3 and T-bet in (b) CD44^hi memory phenotype CD8+ T cells or (c) CD44^lo CD5^hi CD8+ T cells. Cells were stimulated with PMA/Ionomycin or not, as indicated. Using Boolean gating, the average frequency of (stimulated) CD44^hi cells positive for CXCR3, T-bet AND XCL1 was 36.5%, while that for CD44^lo, CD5^hi cells was 1.2% (data not shown) in this experiment. These data are representative of 3 experiments (n=7), utilizing T-bet reporter cells.