Galectin-1 mediated suppression of Epstein-Barr virus specific T-cell immunity in classic Hodgkin lymphoma

Abstract

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg cells interact with the host microenvironment to create an immunosuppressive network that protects the lymphoma from immune attack. These mechanisms are not fully understood. We examined the role of the immunomodulatory protein galectin-1 (Gal-1) on Epstein-Barr virus-specific CD8(+) T cell responses in HL. Initial studies indicated Gal-1 expression in all in vitro established Hodgkin Reed-Sternberg cell lines. In situ analysis revealed Gal-1 expression in 26 of 42 classic HL, whereas Gal-1 was uniformly negative in nodular lymphocyte predominant HL. Gal-1(hi) expression was associated with male gender, older patients, reduced CD8(+) T cell infiltration at the tumor site, and most importantly, an impaired latent membrane protein 1 and 2-specific CD8(+) T-cell responses. In vitro exposure to recombinant Gal-1 inhibited proliferation and interferon-gamma expression by Epstein-Barr virus-specific T cells. These observations provide an important link between the Gal-1-mediated immunomodulatory networks and loss of antigen-specific T-cell function in classic HL.

Figure 1

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.