Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus

Abstract

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

Keywords: T cells; galectin 1; glycosylation; glycosylation enzymes; lectin binding; sialylation; systemic lupus erythematosus.

Figure 1

Lectin binding properties of resting and activated T cells from healthy donors and from systemic lupus erythematosus (SLE) patients. Peripheral blood T cells were obtained from healthy controls and SLE patients. The cells were left unstimulated (resting state, A) or were activated with 1 µg/mL phytohaemagglutinin L (PHA L) for 72 h (activated state, B). Cells were stained with viability dye, fixed then labeled with anti-CD3-PE-Cy5 antibody, followed by fluorescein isothiocyanate (FITC)-conjugated lectin. The samples were evaluated with flow cytometry. Binding of FITC-conjugated lectins is shown as mean (±SEM) of the median fluorescence intensity (MFI) values of flow cytometry histograms of resting (A) or activated (B) CD3-positive live T cells. Lectin names are listed in Table 1. MFI: mean fluorescence intensity, ConA: concanavalin-A, LCA: Lens culinaris agglutinin, WGA: wheat germ agglutinin, PHA-L: Phaseolus vulgaris leukoagglutinin, SNA: Sambucus nigra agglutinin, Gal-1: galectin 1. Statistical analysis was performed using an unpaired Student t-test. ** p < 0.01; *** p < 0.001; n. s.: not significant. SLE: n = 18, and healthy controls: n = 19.

Figure 2

Gene expression of mannosidases (MANs) (A) and N-acetyl glucosaminyltransferases (MGATs) (B) in activated T cells. Total RNA was extracted from activated T cells and mRNA expression levels were analyzed by qPCR. Results of the relative expression were normalized to the expression levels of the RPL27 housekeeping gene (log₂ transformation, ΔCt). Gene names and primer sequences are listed in Table 2 and Table 4, respectively. Upper and lower quartiles and whiskers of boxes extend to the minimum and maximum values, and the band inside the box is the median. Statistical analysis was performed using an unpaired Student’s t-test, where * p < 0.05; SLE: n = 18, and healthy controls: n = 19.

Figure 3

Gene expression of sialyltransferases (ST) and neuraminidase-1 (NEU1) in activated T cells. Total RNA was extracted from activated T cells and mRNA expression levels were analyzed by qPCR. (A) Results of the relative expression were normalized to the expression levels of RPL27 housekeeping gene (log₂ transformation, ΔCt). Gene names and primer sequences are listed in Table 2 and Table 4, respectively. (B) ST/NEU1 mRNA expression ratios. The sialyltransferase-neuraminidase mRNA expression ratios of individual persons were calculated as follows: ΔCt ST/ΔCtNEU1. Upper and lower quartiles and whiskers of boxes extend to the minimum and maximum values, and the band inside the box is the median. Statistical analysis was performed using an unpaired Student t-test, where ** p< 0.01; *** p< 0.001. SLE: n = 18, and healthy controls: n = 19.

Figure 4

Effect of neuraminidase treatment on Gal-1 binding of SLE T cells. Activated SLE T cells were treated with α2-3,6,8 neuraminidase (Gal-1 + α2-3,6,8 Neu; dotted line) or left untreated (Gal-1; empty, continuous line), and then Gal-1 binding was investigated by cytofluorimetry, as described in the materials and methods section. The grey shadowed histogram shows the negative control: no Gal-1, +streptavidin—FITC. The upper image shows a representative profile of Gal-1 binding, the lower graph shows means (±SEM) of the mean fluorescence intensity (MFI) values of activated SLE T cells. Statistical analysis was performed using a two-tailed paired t-test. * p < 0.05, n = 3.