Fig 1: JAM-B does not suffice to arrest CD8 T cells under shear, but it favors their crawling. (a) Number of in vitro activated OT-I cells arrested per field of view (FOV) on recombinant mouse JAM-B (10 µM), VCAM-1 (10 µM), JAM-C (10 µM), or the combination of JAM-B and JAM-C or JAM-B and VCAM-1 (10 µM per molecule) under physiological flow. Recombinant mouse DNER was used as a negative control. Data are shown as mean ± SD and were analyzed by using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. Data are representative of 3 independent experiments, where each experiment represents an individual T cell preparation. (b) Bar graphs depict crawling speed in µm/min of in vitro activated OT-I cells on 10 µM VCAM-1 and increasing concentrations of JAM-B. Values are pooled from six individual movies from 3 independent T cell preparations. Data are shown as mean ± SEM and were analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. For each condition 100–200 cells were tracked. (c) In vivo activated OT-I cells were isolated from the spleen and peripheral lymph nodes of ODC-OVA; JAM-BWT and ODC-OVA; JAM-BKO mice on day 3 after LCMV-OVA infection. Bar graphs depict the crawling speed in µm/min of in vivo activated OT-I cells on VCAM-1 alone or of equimolar concentrations of VCAM-1 and JAM-B. Values are pooled from two individual experiments, where each experiment represent an independent T cell preparation. Data are shown as mean ± SEM and were analyzed using a two-sided parametric T test. For each condition a minimum of 50 cells were tracked.
Fig 2: CD8 T cells do not bind to recombinant JAM-B under static conditions in vitro. Number of in vitro activated OT-I cells (a) and in vivo activated OT-I cells (b-d), bound on slides coated with recombinant JAM-B (10 µM), VCAM-1 (10 µM), and JAM-C (10 µM) or the combination of JAM-B and JAM-C or JAM-B and VCAM-1 (10 µM per molecule) per field of view (FOV). Recombinant mouse DNER-IgG (10 µM) was used as a negative control. a) The bar on the right side shows the number of OT-I cells bound to the combination of recombinant mouse JAM-B and VCAM-1 after OT-I cell preincubation with a blocking antibody against α4-integrin. Data are shown as mean ± SD, analysed by one-way ANOVA with Tukey’s multiple comparisons test. Data are representative of 4 independent experiments performed in triplicates. b-d) In vivo activated OT-I cells were isolated from the spleen and peripheral lymph nodes of ODC-OVA; JAM-BWT (b) and ODC-OVA; JAM-BKO (c) mice on day 3 after LCMV-OVA infection. Bar graph in (d) shows the number of in vivo activated OT-I cells per field of view bound to recombinant mouse VCAM-1. Data are shown as mean ± SD, analysis by one-way ANOVA with Tukey’s multiple comparisons test. Data are representative of 3 independent experiments. e-f) Number of in vitro activated human CD8 (e) and CD4 (f) T cells per field of view bound to recombinant human JAM-B (100 nM), VCAM-1 (100 nM), JAM-C (100 nM), or the combination of JAM-B and JAM-C or JAM-B and VCAM-1 (100 nM per molecule). Data are shown as mean ± SD, analysis by one-way ANOVA with Tukey’s multiple comparisons test. Data are representative of 2 independent experiments in (e) and 1 in (f)
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