Fig 1: Rap1 promotes T cell polarization via RhoA signaling(A) RhoA activation with CCL21 was Gαi dependent. T cells treated with or without pertussis toxin (PTX, 200 ng/mL, 2 h) were stimulated with CCL21 (100 nM) and stained for RhoA-GTP with specific antibodies. Bars indicate the average of median fluorescent intensity (MFI) ± SD (n = 3).(B) RhoA-GTP of WT and Rap1a−/−Rap1b−/−naïve T cells (CD44low) 0, 2, and 5 min after CCL21 stimulation (100 nM). The results are shown as above (n = 3).(C) Relative amount of RhoA-GTP against total RhoA in WT and Rap1a−/−Rap1b−/−naïve T cells (CD44low) with or without CCL21 (100 nM) at 120 s (n = 3).(D) Phosphorylated MLC at Ser 19 (pMLC) in WT and Rap1a−/−Rap1b−/− T cells without or with CCL21 (100 nM). The relative amount of pMLC in mutant T cells was calculated as MLC levels by the MFI normalized to the average of MFI of unstimulated WT T cells. Bars indicate the average ±SD.(E) Impaired clustering of pMLC in Rap1a−/−; Rap1b−/− T cells. Two examples of 3D projection of F-actin and pMLC with composite-colored images (merged) in WT and Rap1a−/−Rap1b−/− T cells stimulated with CCL21 (100 nM).(F) Clustering of pMLC in WT and Rap1a−/−Rap1b−/− T cells. Contour plots of pMLC clustering (x axis) vs. circularity (y axis) with percentages of pMLC clustering cells in the gate. Bar shows average percentage ±SD of pMLC-clustering cells (n = 2–3).(G) RhoA-GTP in WT and Rasa3−/−; Sipa1−/−naïve T cells. Bars indicate average MFI ±SD (n = 3).(H) Relative amount of RhoA-GTP against total RhoA in WT and Rasa3−/−Sipa1−/− naive T cells (CD44low). Bars indicate average ±SD (n = 3).(I) pMLC in Rasa3−/−Sipa1−/− T cells in the absence or presence of Y27632 (20 μM). Bars indicate average ±SD (n = 3, a representative of two independent experiments).(J) Suppression of cell polarization of Rasa3−/−Sipa1−/− T cells with ROCK inhibitor Y27632 (20 μM) and myosin II inhibitor blebbistatin (50 μM). Bars show average percentages ±SD of elongated cells (n = 3).(K) The phosphorylation of NDR1 at Thr444 in WT and Rap1a−/−Rap1b−/− T cells. The right bar graph shows the average amount of NDR1 against total Rap1 (n = 2).(L) Impaired clustering of GEF-H1 in Rap1a−/−Rap1b−/− T cells. 3D projections of GEF-H1, F-actin, and composite images.(M) Changes of GEF-H1 clustering upon CCL21 stimulation (100 nM). Bars indicate the average percentages ±SD (n = 3) of GEF-H1-clustered cells.(N) GEF-H1 clustering of Rasa3−/−Sipa1−/− T cells in the absence of chemokine as shown in (M). Statistical significance for the above data was determined by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 2: Cells migrate under the agarose spot containing a chemokine according to the expression of the cognate chemokine receptor. PC-3 cells which express chemokine receptors CXCR4 and CCR7 migrate under agarose spot containing ligand for CXCR4, CXCL12, (A), and ligands for CCR7, CCL21, (B) and CCL19, (C), but do not migrate under agarose spot containing no chemokine, (D). (E) Relative chemotactic aptitude of PC-3 cells under agarose spot containing 100 nM CXCL12 (ligand for CXCR4), and agarose spot containing 100 nM CCL19 and 100 nM CCL21 (ligands for CCR7), but not under agarose spot containing no chemokine (n = 3). (F) SW480 cells which express chemokine receptor CXCR4 but not CCR7, migrate under agarose spot containing 200 nM CXCL12 (ligand for CXCR4), but not under agarose spot containing 200 nM CCL19 and 200 nM CCL21 (ligands for CCR7), or agarose spot containing no chemokine (n = 3).
Fig 3: Effects of the Itgb2W748A mutation on cell polarization(A) Expression of ICAM1, ICAM2, VCAM1, and MAdCAM1 in WT and Itgb2W748A KI T cells. Red line: isotype control, blue line: ICAM1, ICAM2, VCAM1, and MAdCAM1.(B) Expression of αL and β2 subunits of naive WT and W748A mutant T cells. Red line: isotype control, blue line: αL and β2.(C) Adhesion of WT, Itgb2W748A KI (left panel) and Tln1−/− T cells (right panel) on ICAM1 (n = 3–5).(D) Changes of cell elongation and cell polarization of naive WT, W748A and Tln1−/− T cells after CCL21 stimulation (100 nM) in RPMI1640 containing 1% BSA, measured by Imaging cytometer. Bars represent the average percentage ±SD of elongated cells (n = 3). Statistical significance for the above data was determined by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, NS: not significant.
Fig 4: Defective polarization of T cells deficient in Rap1(A) Expression profiles of CCR7 on WT (red) and Rap1a−/−Rap1b−/− (blue) T cells. CCR7 expression was measured using CCL19-Fc and FITC-conjugated anti-human IgG. A representative histogram of two experiments is shown. Solid and dotted lines represent the bindings of CCL19-Fc and Fc proteins, respectively.(B) A schematic flow of experimental procedures for cell polarity measurement.(C) The typical cell shapes with F-actin and CD44 distribution in unpolarized and polarized cells and representative images acquired using an imaging cytometer as in (B), with numerical values of the aspect ratios, circularity, and polarity. Polarized cells exhibited low aspect ratios and circularity, but high polarity.(D) Contour plots with polarity (x-axis) and circularity (y axis) of WT and Rap1a−/−Rap1b−/−naïve T cells in the absence (upper) and the presence (lower) of CCL21 (100 nM). The numbers show percentages of cells in the gate 5 min after stimulation.(E) The changes in the cell elongation and polarization of naive T cells after the stimulation of CCL21 (100 nM). The data at each time point show the average percentages ±SD (n = 3) of WT (black) and Rap1a−/−Rap1b−/− (red) T cells exhibiting cell elongation (left) and cell polarity (right).(F) Clustering of F-actin (left) and CD44 (right), shown as in (E).(G) Evaluation of defective polarity in Rap1a−/−; Rap1b−/− T cells with supervised machine learning. The score profiles of CCL21-stimulated WT and Rap1a−/−Rap1b−/− T cells as judged by AI were statistically significant (chi-squared test, p < 0.0001) (see STAR Methods).(H) The changes in the cell polarization of CCL21-stimulated WT and Rap1a−/−Rap1b−/− T cells were judged by AI. The average percentages ±SD (n = 3) of polarized (Hand-Mirror score >0.9) cells were shown. Statistical significance for all data was determined by two-tailed Student’s t test except (G), which was determined by a chi-squared test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001.
Fig 5: WBH treatment enhances α4 integrin mediated T cell adhesion and transmigrationC57BL/6J mice were treated with normothermia (NT) or fever-range whole-body hyperthermia (WBH) for 6 h, and then were sacrificed. T cells were isolated from spleen. α4β7–VCAM-1 binding was disrupted by pre-treating the cells with 10 μg/mL α4β7 blocking antibody DATK32 when examining α4β1-mediated cell adhesion and migration on VCAM-1 substrate.(A) Adhesion of T cells to immobilized VCAM-1-Fc (5 μg/mL) or MAdCAM-1-Fc (5 μg/mL) substrate in 1 mM Ca2+ + Mg2+ at a wall shear stress of 1 dyn/cm2.(B) Transmigration of T cells across VCAM-1-Fc (5 μg/mL) or MAdCAM-1-Fc (5 μg/mL) coated membrane in the presence of CCL21 (500 ng/mL) in the lower chamber.Data represent the mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (Student’s t test). The asterisk in (A) indicates the changes in total adherent cells.
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