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Results: 5

1.
Fig. 4.

Fig. 4. From: Mycolactone impairs T cell homing by suppressing microRNA control of L-selectin expression.

Mycolactone impairs T-cell response to antigen in vivo. Purified CD45.1+ CD45.2+ OT-I T cells or CD45.2+ OT-I T cells were stained with CFSE then exposed in vitro to mycolactone (Myco) or vehicle (Ctrl) for 16 h, respectively. Cells were mixed at a ratio of 5:1 (Materials and Methods) and adoptively transferred into CD45.1-recipient mice, which were injected concomitantly into the hind footpad with OVA. (A) A representative CFSE profile is shown for CD45.1+ CD45.2+ (Myco, black) and CD45.2+ (Ctrl, gray) positive LN T cells 48 h after immunization, with peak labels indicating the successive generations. (B) Mean percent dividing cells ± SEM in each cycle, calculated from four mice. Independent experiments performed with Myco and Ctrl cells delivered at a ratio of 1:1 gave comparable results. **P < 0.01.

Laure Guenin-Macé, et al. Proc Natl Acad Sci U S A. 2011 August 2;108(31):12833-12838.
2.
Fig. 1.

Fig. 1. From: Mycolactone impairs T cell homing by suppressing microRNA control of L-selectin expression.

Mycolactone induces lymphocyte depletion in PLNs. (A) Macroscopic view of inguinal LNs of mice 24 h after s.c. injection of 50 or 100 μg of mycolactone, or vehicle as control, at the base of the tail. (B) Hematoxilin/eosin (a), B220 + CD3 (b), PNAd + CD3 (c), and ER-TR7 + CD3 (d) staining of PLNs of mice injected with 50 μg of mycolactone (Lower) or vehicle as control (Upper). Photos in A and B are representative of at least two independent experiments. (C) CD4+ and CD8+ T cells counts in inguinal LNs and peripheral blood of mice 24 h after s.c. injection of 100 μg of mycolactone or vehicle (Ctrl). Data are mean cell counts ± SEM of two independent experiments using three mice per group.

Laure Guenin-Macé, et al. Proc Natl Acad Sci U S A. 2011 August 2;108(31):12833-12838.
3.
Fig. 3.

Fig. 3. From: Mycolactone impairs T cell homing by suppressing microRNA control of L-selectin expression.

Mycolactone-treated T cells have impaired homing capacity and reduced motility response to CCR7. (A) T cells purified from mouse PLNs were cultured in the presence of 100 ng/mL mycolactone (Myco) or vehicle (Ctrl) for 16 h, loaded with two different concentrations of CMFDA, then mixed in equal numbers and adoptively transferred into recipient mice. Total counts of mycolactone-treated T cells are compared with that of controls (100%) in PLNs and spleen of recipients 3.5 h after transfer. Data are mean cell counts ± SEM from three experiments using three recipient mice. (B) PLN T cells were incubated for 16 h with 100 ng/mL mycolactone (Myco) or vehicle (Ctrl), then labeled with two different fluorescent dyes and overlaid on LN slices. Data are mean motility and velocity coefficients ± SEM from three independent experiments, with >100 cells analyzed per experiment. *P < 0.05; **P < 0.01; NS, not significant.

Laure Guenin-Macé, et al. Proc Natl Acad Sci U S A. 2011 August 2;108(31):12833-12838.
4.
Fig. 2.

Fig. 2. From: Mycolactone impairs T cell homing by suppressing microRNA control of L-selectin expression.

Mycolactone modulates the expression of T-cell homing receptors in vivo and in vitro. (A) Kinetic analysis of the CD62-L expression and concentration of blood CD4+ and CD8+ T cells after the s.c. injection of 100 μg of mycolactone. Data are mean fluorescence intensities (MFI) and mean cell counts ± SEM from three mice, expressed as percent of controls. They are representative of two independent experiments. (B) Expression of CD62-L, CCR7, and LFA-1 on CD4+ and CD8+ PLN T cells of mice injected with 100 μg of mycolactone or vehicle (Ctrl). Data are MFI ± SEM measured 16 h after s.c. injection from five mice, expressed as percent of controls. (C) Expression of CD62-L, CCR7, and LFA-1 on PLN T cells cultured for 16 h in the presence of 100 ng/mL mycolactone, or vehicle (Ctrl). Data are MFI ± SEM of triplicates, expressed as percent of controls. Experiments in B and C were repeated three times independently with comparable results. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Laure Guenin-Macé, et al. Proc Natl Acad Sci U S A. 2011 August 2;108(31):12833-12838.
5.
Fig. 5.

Fig. 5. From: Mycolactone impairs T cell homing by suppressing microRNA control of L-selectin expression.

Mechanism of mycolactone-induced suppression of CD62-L. (A) Time-dependent modulation of CD62-L total and surface expression, as measured by flow cytometry on permeabilized or nonpermeabilized cells, by CD4+ T cells exposed to 100 ng/mL mycolactone or vehicle. Data are MFI ± SEM of triplicates, expressed as percent of controls, and are representative of >3 donors. (B) Effect of mycolactone on CD62-L and KLF2 transcript abundance in CD4+ T cells. Data are mean fold changes ± SEM from three donors, after cell treatment with 100 ng/mL mycolactone, compared with controls by random effect models (Materials and Methods). (C) Effect of mycolactone on the expression of let-7b by T cells. Data are mean fold changes ± SEM from Jurkat cells (n = 4) or PBLs (three donors) treated with 100 ng/mL mycolactone for 30 min or 4 h, respectively, compared with controls. (D) Expression of CD62-L mRNA in PBLs transfected with an anti-let-7b or an irrelevant anti-miR-Ctrl for 24 h. Data are mean fold changes ± SEM from three donors. (E) Expression of CD62-L mRNA in PBLs transfected with pre-Mir-let-7b for 24 h (let-7b), then incubated with 100 ng/mL mycolactone for 4 h (Myco + let-7b). Controls are cells transfected with an irrelevant premiR for 24 h, then incubated with mycolactone (Myco) or vehicle (Ctrl). Data are fold changes from >5 donors, compared with premiR-transfected controls by Wilcoxon matched pairs signed rank sum test, presented as box and whiskers. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Laure Guenin-Macé, et al. Proc Natl Acad Sci U S A. 2011 August 2;108(31):12833-12838.

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