Galectin-1 (Gal-1) expression in Hodgkin lymphoma (HL) cell lines and human melanoma cell line.(A) Western blot analysis for galectin-1 (Gal-1) expression in Hodgkin lymphoma (HL) cell lines and human melanoma cell line: SKMa128 using an anti-Gal-1 antibody. Recombinant Gal-1 was used as a positive control. Lane 1: L428, lane 2: L540, lane 3: L1236, lane 4: HDLM2, lane 5: recombinant Gal-1, and lane 6: SKMel28. (B) Representative photomicrographs showing immunohistochemical staining with anti-Gal-1 or isotype control antibody in a mixed cellularity HL tumor biopsy. Hodgkin Reed-Sternberg cell shows strong Gal-1 expression most marked within the cytoplasmic rim. (Left panel) Original magnification ×40/.65 NA; (right panels) original magnification ×10/.25 NA using a CX41 (Olympus, Tokyo, Japan). Photomicrographs were taken using a Nikon Coolpix 5700 (Tokyo, Japan) and Microsoft Office XP Photo Editor (Seattle, WA) used for image acquisition. (C) Effect of Gal-1 expression on the CD8⁺ T cell infiltration in the tumor tissue. Data are presented as mean (± SE) of the number of infiltrating CD8⁺ T cells within Hodgkin Reed-Sternberg-rich areas in Gal-1^hi and Gal-1^lo HL patients. Asterisk denotes statistical significance (P = .048). (D) Effect of Gal-1 expression on Epstein-Barr virus (EBV)-specific CD8⁺ T cell response. Data are presented as a mean (± SE) of ex vivo EBV-specific interferon-γ spot-forming cells/10⁶ peripheral blood mononuclear cells after stimulation with HLA class I-restricted epitopes from EBV proteins (LMP1 and 2: left panel; EBNA3/4/6 and lytic: right panel). All T cell responses were assessed before initiation of therapy, and the data were subdivided into Gal-1^hi and Gal-1^lo HL patients. Asterisk denotes statistical significance (P = .046). (E) Effect of recombinant Gal-1 on the proliferation of EBV-specific T cells. Peripheral blood mononuclear cells from EBV-seropositive patients were labeled with carboxy fluorescein diacetate succinimidyl ester, preincubated with Gal-1 at 1 μg/L (or mock-treated), and stimulated with autologous lymphoblastoid cell line at 20:1 responder: stimulator ratio. CD8⁺ T cell proliferation was assessed after 7 days culture. Results are representative of 5 separate experiments. (F) Effect of recombinant Gal-1 on the expression of interferon-γ by EBV-specific CD8⁺and CD4⁺ T cells. Peripheral blood mononuclear cells from EBV-seropositive patients were preincubated with Gal-1 at 1 μg/L (or mock-treated) and then stimulated with autologous lymphoblastoid cell line at different responder:stimulator ratios. Interferon-γ expression by EBV-specific CD8⁺, CD4⁺, and LMP2-specific CD8⁺ T cells were assessed using intracellular cytokine assay. LMP2-specific T cells were stained with APC-labeled major histocompatibility complex-peptide pentamer for HLA A2-restricted epitope CLGGLLTMV. Data are presented as relative interferon-γ expression by antigen-specific T cells with respected to the mock-treated cells. Results are representative of 2 separate experiments.