Fig 2: CCL5+ CTCs are positively correlated with circulating Tregs and predicted postoperative relapse in HCC patients.a Immune-evasion-related genes and cytokines differentially expressed by CTCs and primary tumors. b Multiplex immunofluorescence images displaying the expression of CCL5 in primary tumors and CTCs detected in peritumoral microvasculature. mVI, microvascular invasion. The scale bars represent 20 µm, 200 µm, and 5 µm, respectively. c Multiplex immunofluorescence images representative of spatial relationship between the CCL5+ CTCs and CCR5+/FoxP3+ Tregs detected in peritumoral microvasculature. The scale bars represent 200 µm and 10 μm, respectively. d Scatterplot showing a positive correlation between the number of CCL5+ CTCs and the abundance of CCR5+ Tregs (CD4+, CD25high, and CD127low) (upper) and total Tregs (lower) in CD4+ T cells from HCC peripheral blood (n = 27 patients). e Kaplan–Meier analysis showing increased probability of early recurrence (left) and decreased overall survival rate (right) in patients with Treghigh/CTChigh in peripheral blood vs the other groups. I: Treglow/CCL5+ CTClow, II: Treglow/CCL5+ CTChigh, III: Treghigh/CCL5+ CTClow, and IV: Treghigh/CCL5+ CTChigh. The number of patients at risk for each group is listed below the Kaplan–Meier curve. A two-tailed Student’s t test was employed (d). Log-rank testing are performed to estimate the prognostic significance (e).

Fig 3: LPS injected intraperitoneally induces RAT of CCR5 in macrophages.Macrophages isolated from peritoneal cavity of mice injected intraperitoneally with 1 mg/kg LPS and 1 mg/kg FB1 for 16 hr were analyzed as described in Figure 3B–I. Scale bar = 20 µm.

Fig 4: RAT of CCR5 blocks migration of macrophages toward CCL5.(A) Migration of macrophages from mice with indicated genotype was assayed in transwell plates for 24 hr with or without 100 ng/ml LPS in the upper wells and 100 nM CCL5 as the chemoattractant in lower wells. Signals generated in the absence of CCL5 were subtracted to normalize for random migration. (B) Migration assays were performed as described in (A) with wildtype macrophages incubated with LPS and 30 µM FB1 in upper wells as indicated. (A and B) Results were reported as mean ±S.E. from three independent experiments, with the value obtained from untreated WT macrophages set at 100%.

Fig 5: LPS induces RAT of CCR5 in primary mouse macrophages.(A–I) Mouse macrophages were treated with or without 100 ng/ml LPS in the absence (B–E) or presence of 30 µM FB1 (F–I) for 16 hr. (A) The amount of dihydroceramide with the indicated amide-linked acyl chains in the cells was determined through LC-MS measurement. Results are reported as mean ±S.E. from three independent experiments. (B–I) Macrophages were subjected to immunofluorescent microscopy analysis with an antibody against the seoncd extracellular loop of CCR5(A) in the absence or presence of saponin-mediated cell permeabilization. Scale bar = 10 µm. (J and K) Macrophages treated without (J) or with (K) 100 ng/ml LPS for 24 hr were subjected to immuno-gold EM analysis with an antibody against the N-terminal domain of CCR5. Scale bar = 200 nm. (L) The number of intracellular and extracellular-localized CCR5 labeled by gold clusters per cell was quantified from macrophages treated with (n = 20) and without LPS (n = 28). The results are reported as mean ±S.D. This number should be smaller than that of CCR5 molecules, as it did not include CCR5 labeled by a single gold particle, the specificity of which was difficult to determine.