Galectin-1 inhibits the viability, proliferation, and Th1 cytokine production of nonmalignant T cells in patients with leukemic cutaneous T-cell lymphoma

Abstract

Tumor-derived galectin-1 (Gal-1), a β-galactoside-binding S-type lectin, has been shown to encourage T-cell death and promote T cell-mediated tumor immune escape. In this report, we show that patients with leukemic cutaneous T-cell lymphomas, known to have limited complexity of their T-cell repertoires, have a predominant T helper type-2 (Th2) cytokine profile and significantly elevated plasma levels of Gal-1 compared with healthy controls. Circulating clonal malignant T cells were a major source of Gal-1. The conditioned supernatant of cultured malignant T cells induced a β-galactoside-dependent inhibition of normal T-cell proliferation and a Th2 skewing of cytokine production. These data implicate Gal-1 in development of the Th2 phenotype in patients with advanced-stage cutaneous T-cell lymphoma and highlight the Gal-1-Gal-1 ligand axis as a potential therapeutic target for enhancing antitumor immune responses.

Figure 1

Patients with L-CTCL exhibit a contracted T-cell repertoire and Th2 cytokine profile. (A) PBMCs from patients with stage 4 CTCL or healthy controls (n = 3/group) were analyzed by flow cytometry. Representative plots show cells analyzed for CD3 and CD4 expression. Arrow indicates contraction of the CD3⁺/CD4⁻ population. (B) Th cells from patients with stage 4 CTCL show increased expression of a single malignant TCR-Vβ clone. Representative plot is shown. (C) PBMCs from healthy controls or L-CTCL patients (n = 3/group) were stimulated ex vivo with PMA/ionomycin/brefeldin A, and intracellular levels of Th2 cytokines were analyzed by flow cytometry. Representative plot of L-CTCL patients is shown. (D) Graphical representation of data from all L-CTCL and health control donors depicting mean percentage of CD4⁺ cells expressing IL-4, IL-13, and IL-10. Statistically significant difference compared with healthy controls using Student paired t test: *P ≤ .05, **P ≤ .001. (E) PBMCs from healthy controls or L-CTCL patients were stimulated ex vivo with PMA/ionomycin/brefeldin A, and intracellular levels of IL-10 were analyzed in CD8⁺ T cells by flow cytometry. A representative plot is shown. (F) Graphical representation of undiluted plasma samples from patients with advanced-stage CTCL (triangles, n = 7) and healthy controls (squares, n = 5) were analyzed for Gal-1 expression by ELISA. Experiments were performed in triplicate. (G) PBMCs from L-CTCL patients or healthy controls were gated on CD3⁺/CD4⁺ and analyzed for specific TCR-Vβ expression and intracellular Gal-1. A representative plot is shown.

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-1^hi 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.