Figure 1. Differential KLF2 and S1PR1 expression by memory CD8⁺ T cells in lymphoid and non-lymphoid tissues.

(a) KLF2^GFP expression in splenic memory-phenotype CD8+ T cells (representative of n=9 from 3 independent experiments). (b) Expression of KLF2^GFP in antigen-specific CD8⁺ T cells (endogenous D^b/gp33 tetramer⁺ or adoptively transferred P14 CD8⁺ T cells) from indicated tissues, >28 days after LCMV infection. Data show fluorescence of KLF2^GFP CD8⁺ T cells (black line) overlaid on CD8⁺ T cells from matched WT controls (grey filled). Data are representative of 6–12 animals from at least 3 independent experiments. (c, d) Analysis of KLF2^GFP expression in adoptively transferred P14 CD8⁺ T cells, >28 days post LCMV infection. (c) IV anti-CD8 antibody was used to distinguish P14 T cells in tissue parenchyma (solid line) versus vascular-associated cells (dashed line). Data are overlaid with WT P14 CD8⁺ T cells (grey filled) as controls (representative of n=9 from 3 independent experiments). (d) CD69 versus KLF2^GFP expression for parenchymal P14 T cells within indicated tissues (representative of n=9 from 3 independent experiments). (e) RNA was isolated from sorted P14 CD8⁺ T cells, isolated from spleen, salivary glands and LPL 30 days post LCMV infection, and subjected to RT-PCR for the indicated genes. Gene expression (relative to HPRT control) was normalized to the spleen for comparison between experiments. Compiled from four independent experiments (9–12 pooled mice each experiment), graphs show mean +/− SD. (f) Activated P14 CD8⁺ T cells were transduced with retroviral vectors encoding KLF2 (or a control, “Empty” vector). Two days later, cells expressing the transduction marker (Thy-1.1) were enriched and subjected to RT-PCR for the indicated genes. Gene expression, relative to HPRT controls, was normalized on empty vector controls. Data are compiled from 3 separate transduction experiments. At the same time point, cell surface expression of CD69 was determined on Thy-1.1+ve transduced cells (inset) - data shown are representative of at least 3 transduction experiments. Statistical analysis utilized one-way ANOVA (with Dunnett’s Multiple Comparison Test in (e)) and significance is indicated as follows: ***, p<0.001; **, p<0.01; *, p<0.05; NS, p>0.05.