Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry

Abstract

Interaction of cell surface glycoproteins with endogenous lectins on the cell surface regulates formation and maintenance of plasma membrane domains, clusters signaling complexes, and controls the residency time of glycoproteins on the plasma membrane. Galectin-9 is a soluble, secreted lectin that binds to glycoprotein receptors to form galectin-glycoprotein lattices on the cell surface. Whereas galectin-9 binding to specific glycoprotein receptors induces death of CD4 Th1 cells, CD4 Th2 cells are resistant to galectin-9 death due to alternative glycosylation. On Th2 cells, galectin-9 binds cell surface protein disulfide isomerase (PDI), increasing retention of PDI on the cell surface and altering the redox status at the plasma membrane. Cell surface PDI regulates integrin function on platelets and also enhances susceptibility of T cells to infection with HIV. We find that galectin-9 binding to PDI on Th2 cells results in increased cell migration through extracellular matrix via β3 integrins, identifying a unique mechanism to regulate T-cell migration. In addition, galectin-9 binding to PDI on T cells potentiates infection with HIV. We identify a mechanism for regulating cell surface redox status via a galectin-glycoprotein lattice, to regulate distinct T-cell functions.

Fig. 1.

Galectin-9 binds to T-cell surface PDI. (A) Galectin-9 binds T cells. PhaRST6 cells were treated with PBS (thin line), 0.1 μM biotin–galectin-9 (thick line), or 0.1 μM biotin–galectin-9 plus 100 mM lactose (dotted line), and bound galectin-9 detected with 0.1 μg streptavidin-PE. (B) Isolation of T-cell glycoproteins that bind galectin-9. T-cell membrane proteins bound to a galectin-9 affinity column were eluted with lactose and stained with SYPRO Ruby. The band (*) was determined to be PDI (P4HB) by mass spectrometry. (C) Reactivity with anti-PDI mAb RL77 confirmed PDI in eluate from galectin-9 affinity column. (D) Cell surface PDI coprecipitates with galectin-9. After plasma membrane biotinylation and addition of galectin-9, PhaRST6 cell extracts were treated with IgG control (Ctrl) or anti–galectin-9 (Ab) and bound material precipitated with protein G sepharose. Precipitates were probed with streptavidin-HRP, polyclonal anti-PDI, or anti-PDI mAbs RL77 or RL90. (E) PDI is abundantly expressed on PhaRST6 and murine Th2 cells, but not Th1 cells; thick line, anti-PDI; thin line, isotype control. α2,6-linked sialic acid, detected with SNA, is abundant on PhaRST6 and murine Th2 cells, compared with Th1 cells; thick line, biotinylated SNA; thin line, biotinylated BSA. (F) Galectin-9 binding increases abundance of cell surface PDI. 0.1 μM galectin-9 was added to PhaRST6 and murine Th2 cells and cell surface PDI detected by flow cytometry: thin line, IgG control; bold line, anti-PDI on control cells; gray solid, anti-PDI on galectin-9 treated cells. (G) 0.1 μM galectin-9 increased abundance of free thiols, detected with Alexa Fluor 488 C5 maleimide on PhaRST6 and murine Th2 cells; results are mean ± SE of five experiments for PhaRST6 cells and three experiments for Th2 cells, each in triplicate, shown as fold-change in mean fluorescence intensity (MFI) compared with control. **P < 0.0025. (H) Galectin-9–mediated increase in cell surface PDI requires O-glycans. (Left) Jurkat E6-1 cells were treated without (thick line) or with (thin line) 2 mM benzyl-α-GalNAc for 72 h and phenotyped with biotinylated PNA (dotted line, biotinylated BSA) to demonstrate loss of cell surface O-glycans. (Right) Treatment with benzyl-α-GalNAc reduced galectin-9 (0.1 μM)–mediated increase in cell surface PDI (dotted line, no galectin-9; thin line, galectin-9 on benzyl-α-GalNAc–treated cells; thick line, galectin-9 on control cells). (I) PNA reactivity indicates O-glycans on PDI. Biotinylated PNA was added to Jurkat T-cell lysates, bound glycoproteins precipitated, and probed with anti-PDI mAbs RL77 and RL90.

Fig. 2.

Galectin-9 enhances Th2 and PhaRST6 T-cell migration. (A) Migration of PhaRST6 and Th2 through Matrigel was enhanced by galectin-9 in a dose-dependent fashion. (B) Increased T-cell migration was phenocopied by inclusion of 2-ME (6 mM) in Matrigel. (C) Migration of PhaRST6 cells through Matrigel was only enhanced when galectin-9 was added directly to Matrigel, but not when 0.1 μM galectin-9 was added to the bottom well of the migration chamber. Results are mean ± SE of three experiments, each in triplicate. **P < 0.025.

Fig. 3.

Increased T-cell migration mediated by galectin-9 is PDI dependent. Galectin-9 (0.1 μM) enhanced migration of PhaRST6 cells (A and B) and Th2 cells (C and D) through Matrigel was abrogated by 1:10,000 anti-PDI (α-PDI), 3 mM bacitracin (Bac), and thiol scavengers 0.03 mM tocinoic acid (Toc) and 0.01 mM DTNB. Inhibitors reduced migration in a dose-dependent manner; indicated doses are shown. Results are mean ± SE of three experiments, each in triplicate. *P < 0.01, **P < 0.0025.

Fig. 4.

Galectin-9 enhancement of T-cell migration involves CD61. (A Upper) CD61 (thick line) is expressed on PhaRST6 cells; isotype control (thin line). (Lower) PDI associates with CD61 on PhaRST6 cells; T-cell membrane extracts were immunoprecipitated with IgG control (Ctrl) or anti-CD61 mAb HMbeta3.1 (α-CD61), and precipitate probed with anti-PDI mAb RL77. (B Upper) CD61 (thick line) is expressed on human Th2 cells; isotype control (thin line). (Lower) PDI associates with CD61 on human Th2 cells; T-cell membrane extracts were immunoprecipitated with IgG control (Ctl) or anti-CD61 mAb Y2/51 (α-CD61), and precipitate probed with anti-PDI mAb RL77. (C) Galectin-9 (0.1 μM) enhancement of T-cell migration was abrogated by anti-CD61 mAb; a representative dose (1:10,000 for PhaRST6 cells, 1:30,000 for Th2 cells) is shown. Results are mean ± SE of three experiments, each in triplicate. *P < 0.01.

Fig. 5.

Galectin-9 enhancement of HIV-1 infection is PDI dependent. (A) PDI is expressed on Jurkat cells, and PDI is increased by addition of galectin-9 in a dose-dependent manner. Cell surface staining with IgG control (light gray filled) or anti-PDI without (dark gray filled) or with 0.01 μM (dotted line), 0.03 μM (thin line), or 0.1 μM (bold line) galectin-9 for 2 h. (B) PDI abundance on Jurkat cells was increased by 0.1 μM galectin-9 in a time-dependent manner; mean fluorescence intensity (MFI) ± SD from triplicate samples is shown. (C) Jurkat cells were infected with NL4-3 (CXCR4-tropic HIV-1 strain) at high (20 ng p24) or low (5 ng p24) viral input for 2 h. Galectin-9 (0.1 μM) with or without 3 mM bacitracin (Bac) or galectin-1 (20 μM) was added; after 2 h, cells were washed. At 48 h postinfection, the number of infected cells was quantified by intracellular viral p24 antigen staining detected by flow cytometry. Data from one of three experiments is shown. (D) Galectin-9 enhancement of HIV infection differs from galectin-1. Infection was performed as in C, except that viral entry was quantified by qPCR of early RT products at 6–8 h postinfection. Early RT products were normalized to β-actin levels for each condition, and the number of early RT products in the absence of galectin-9 was set at 1. Galectin-9 enhancement of HIV infection increases over 2 h, the same timeframe in which cell surface PDI increase is seen (B). Galectin-1 enhancement of HIV infection is optimal at 15 min. Results are shown as fold enhancement over HIV-1 alone. (E) Galectin-9 enhancement of HIV infection requires PDI activity. Infection of Jurkat cells was performed as in D using 5 ng (p24 equivalent) of viral inoculum. Results are shown as fold enhancement over HIV-1 alone. Striped bar, no galectin-9; solid bars, 0.1 μM galectin-9; B, 3 mM bacitracin; P, anti-PDI mAB RL77 1:3000; D, 0.01 mM DTNB; T, 0.03 mM tocinoic acid; C, DMSO as vehicle control. Results are mean ± SE of three to five replicate experiments, each done in duplicate.