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. 2013 Nov 18;8(11):e80500. doi: 10.1371/journal.pone.0080500

Figure 3. In vivo migratory responses are impaired in mDia1-deficient mice.

Figure 3

(A) Effector T cells were generated by ex vivo stimulation of Ova-specific T cells from mDia1-/- OT-II or OT-II TCR mice (CD45.2+) with Ova peptide-loaded splenocytes for 6 days and the effector cells then injected into B6.SJL recipient mice (CD45.1) followed by intradermal challenge with OVA-peptide in incomplete Freund’s adjuvant. The accumulation of donor CD45.2 mDia1-/- and wild-type effector T cells in Ova antigen-challenged skin was determined by immunostaining of single skin cell suspensions with PE-conjugated-anti-CD45.2 and Apc-conjugated-anti-CD4 antibodies. (B-G) Intranodal T cells migration. Naive CD4+ T cells purified from the spleens of mDia1-/- and wild-type mice were labeled with CFSE or CMTMR and the mixture (1:1) of CFSE-labeled mDia1-/- and CMTMR-labeled wild-type T cells intravenously injected into C57BL6 mice. Lymph nodes from recipient mice were imaged 24 hrs later by time-lapse two-photon confocal microscopy. The migratory parameters of migrating T cells in time were calculated from the movies using Velocity software. (B) Representative three-dimensional tracks of mDia1-/- and wild-type T cells over a 20 min period. Each colored line represents a single T cell track. (C) Relative frequency of average velocity calculated from the net distance traveled during each time interval (displacement/time during a single time step) of a cell over 20 min; (D) Frequency of mean turning angles from which a cell deviates between successive time steps over 20 min; (E) Frequency of mean square displacement (left panel),a measure of the average distance a given particle in a system travels, and average mean square displacement over 20 min (right panel) are shown for wild-type and mDia1-/- cells. (F) Mean meandering index, a measurement of directionality, was calculated by dividing the distance a cell traveled by the track length. (G) Motility coefficient (M) of mDia1-/- and control T cells. M is a measure for how fast cells displace from their starting positions during a random walk process, analogous to the diffusion coefficient for Brownian motion (M=displacement2/6t). Data are expressed as the mean values ±SEM and are representative of at least 4 independent experiments. ∗∗ indicates p<0.01, *** indicates p<0.001.