Figure 2
Gal-1 secreted from L-CTCL cells dampens T-cell proliferation and promotes Th2 cytokine skewing. (A) Intracellular Gal-1 expression in Th cells from patients with L-CTCL or healthy controls (n = 3/group) was analyzed by flow cytometry and presented as a contour plot. Arrows indicate Gal-1 high, intermediate, and low (“lo”) populations. (B) Graphical representation of intracellular Gal-1 levels quantified by mean fluorescence intensity (MFI). Statistically significant difference compared with healthy controls using Student paired t test. (C) Graphical representation of Gal-1hi Th cells in stage 4 CTCL patients or healthy controls. Statistically significant difference compared with healthy controls using Student paired t test. (D) PBMCs from healthy donors were gated on CD8+, and expression of Gal-1 ligands (Gal-1hFc binding) on CD45RO+ memory cells was determined by flow cytometry. Coexpression of CD7 in CD45RO+/− CD8+ T cells was also analyzed. Representative plots are shown. (E) Activated T cells from healthy donors (n = 3) were incubated with conditioned medium from primary L-CTCL cultures (with or without lactose) for 24 hours and then analyzed for annexin V binding. Statistically significant difference compared with lactose-treated control using Student paired t test: **P ≤ .001. (F) Alternatively, activated T cells were labeled with CFSE, incubated with L-CTCL–conditioned medium (with or without lactose) or with 0.25μM Gal-1hFc (with or without lactose) for 3 days, and CFSE dilution was analyzed by flow cytometry. Activated T cells from healthy donors (n = 3) were incubated with conditioned medium from primary L-CTCL cell cultures (with or without lactose; G) or with 0.25μM Gal-1hFc (with or without lactose; H) for 24 hours and then stained and analyzed for CD3 and intracellular IFN-γ, IL-4, IL-10, and IL-13. All experiments were performed with L-CTCL–conditioned medium from 3 different donors and performed in triplicate. Statistically significant difference compared with lactose-treated control using Student paired t test: *P ≤ .05, **P ≤ .001.

Gal-1 secreted from L-CTCL cells dampens T-cell proliferation and promotes Th2 cytokine skewing. (A) Intracellular Gal-1 expression in Th cells from patients with L-CTCL or healthy controls (n = 3/group) was analyzed by flow cytometry and presented as a contour plot. Arrows indicate Gal-1 high, intermediate, and low (“lo”) populations. (B) Graphical representation of intracellular Gal-1 levels quantified by mean fluorescence intensity (MFI). Statistically significant difference compared with healthy controls using Student paired t test. (C) Graphical representation of Gal-1hi Th cells in stage 4 CTCL patients or healthy controls. Statistically significant difference compared with healthy controls using Student paired t test. (D) PBMCs from healthy donors were gated on CD8+, and expression of Gal-1 ligands (Gal-1hFc binding) on CD45RO+ memory cells was determined by flow cytometry. Coexpression of CD7 in CD45RO+/− CD8+ T cells was also analyzed. Representative plots are shown. (E) Activated T cells from healthy donors (n = 3) were incubated with conditioned medium from primary L-CTCL cultures (with or without lactose) for 24 hours and then analyzed for annexin V binding. Statistically significant difference compared with lactose-treated control using Student paired t test: **P ≤ .001. (F) Alternatively, activated T cells were labeled with CFSE, incubated with L-CTCL–conditioned medium (with or without lactose) or with 0.25μM Gal-1hFc (with or without lactose) for 3 days, and CFSE dilution was analyzed by flow cytometry. Activated T cells from healthy donors (n = 3) were incubated with conditioned medium from primary L-CTCL cell cultures (with or without lactose; G) or with 0.25μM Gal-1hFc (with or without lactose; H) for 24 hours and then stained and analyzed for CD3 and intracellular IFN-γ, IL-4, IL-10, and IL-13. All experiments were performed with L-CTCL–conditioned medium from 3 different donors and performed in triplicate. Statistically significant difference compared with lactose-treated control using Student paired t test: *P ≤ .05, **P ≤ .001.

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