Figure 1

Intravital imaging reveals high motility of naïve CD4+ T cells in the inflamed CNS. Naïve OT2 EGFP T cells were intravenously injected into EAE affected mice at the peak of disease (clinical score 2.5) or locally applied onto the imaging field. Intravital TPLSM on the brain stem of these mice was performed 12 - 24 hours after naïve T cell injection or 30 minutes after local application. Adoptive EAE was induced by transfer of in vitro generated encephalitogenic 2d2 Th17 T cells into C57BL/6 RAG1-/- mice. (A) T cell phenotype of MACS isolated naïve T cells was confirmed by FACS analysis prior to experiments. Surface antigen expression of CD62L, CD25, CD69 and CD44 was determined on CD4+ lymphocytes. (B) A representative time lapse series derived from intravital TPLSM demonstrates rapid movement of naïve T cells deep in CNS tissue (100-150 μm). Two cell tracks are shown exemplarily by white arrows. For further details see also Additional File 1 (Scale bar: 10 μm.). (C) Cell track velocities of naïve OT2 (N = 212) and effector 2d2 Th17 (N = 87) cells at the peak of disease in the inflamed CNS were quantified. The mean track velocities from 4 independent experiments are shown (± SD). (D) Contacts (arrowheads) between encephalitogenic 2d2 Th17 effector T cells (EGFP,green) and naïve OT2 (tdRFP red) could be observed during intravital TPLSM, as revealed by the reconstructed 3D time lapse series (80-110 μm). These interactions were mainly short and random like (open arrowhead) although some static long-lasting contacts (filled arrowhead) could be also detected. (E) To quantify effector-naïve T cell interactions we analyse the co-localisation area of EGFP and tdRFP as previously described [19]. We discriminated short (random) contacts (< 5 min) from long-lasting (most probably non-random) interactions (≥ 5 min) and observed that 81% ± 14 formed short interactions with effector T cells (white bar) and 19% ± 14 (gray bar) formed long-lasting interactions. Data are shwon as percentage of all contacts from two independent experiments (± SD).