Ganglioside GM1 deficiency in effector T cells from NOD mice induces resistance to regulatory T-cell suppression

Abstract

Objective: To detect GM1 deficiency and determine its role in effector T cells (Teffs) from NOD mice in establishing resistance to regulatory T-cell (Treg) suppression.

Research design and methods: CD4(+) and CD8(+) Teffs were isolated from spleens of prediabetic NOD mice for comparison with similar cells from Balb/c, C57BL/6, and NOR mice. GM1 was quantified with thin-layer chromatography for total cellular GM1 and flow cytometry for cell-surface GM1. Suppression of Teff proliferation was determined by application of GM1 cross-linking agents or coculturing with Tregs. Calcium influx in Teffs was quantified using fura-2.

Results: Resting and activated CD4(+) and CD8(+) Teffs of NOD mice contained significantly less GM1 than Teffs from the other three mouse strains tested. After activation, NOD Teffs resisted suppression by Tregs or GM1 cross-linking agents in contrast to robust suppression of Balb/c Teffs; this was reversed by preincubation of NOD Teffs with GM1. NOD Teffs also showed attenuated Ca(2+) influx via transient receptor potential channel 5 (TRPC5) channels induced by GM1 cross-linking, and this, too, was reversed by elevation of Teff GM1.

Conclusions: GM1 deficiency occurs in NOD Teffs and contributes importantly to failed suppression, which is rectified by increasing Teff GM1. Such elevation also reverses subthreshold Ca(2+) influx via TRPC5 channels, an essential aspect of suppression. Our results also support a critical role for galectin-1 as a GM1 cross-linking counter-receptor that fittingly is upregulated and released by Tregs during activation. These findings suggest a novel mechanism by which pathogenic Teffs evade regulatory suppression, thereby leading to autoimmune β-cell destruction and type 1 diabetes.

FIG. 1.

Ganglioside deficiency in NOD Teffs. CD4⁺ and CD8⁺ Teffs were isolated from spleens of 8-week-old NOD and Balb/c mice and activated with anti-CD3/anti-CD28. Lipid extracts from freshly purified (resting) and activated cells were resolved by HPTLC and GM1 family gangliosides revealed by N’ase treatment, followed by CtxB-HRP overlay. A: Resting NOD and Balb/c Teffs (20 μg protein). B: Activated NOD and Balb/c Teffs (10 μg protein). C: Resting NOD, NOR, and C57BL/6 Teffs (20 μg protein). D: Activated NOD, NOR, and C57BL/6 Teffs (10 μg protein). Bovine brain ganglioside (BBG) mixtures of varying amounts were run simultaneously to provide standard curves for quantification (A and B). Resting and activated NOD Teffs both possessed significantly less GM1 and GD1a than Balb/c, NOR, and C57BL/6 Teffs. Note the significant increases of Teff GM1 upon activation. Densitometry results are given in Table 1. Note that in C, lanes 4 and 5 were cut out from the original TLC blot and switched (as designated by two black lines) in order to preserve the order NOD, NOR, C57BL/6.

FIG. 2.

Reduced GM1 (a) and GD1a (b) on the surface of NOD Teffs. NOD and Balb/c CD4⁺ and CD8⁺ Teffs, both resting and activated, were separately stained with CtxB-FITC and anti-GD1a mAb plus anti-mouse IgG-FITC. A: Phase contrast and fluorescent photomicrographs are shown of activated Teffs prepared from 8-week-old mice. B: Flow cytometry shows quantitative deficiencies of surface GM1 and GD1a in resting and activated CD4⁺ and CD8⁺ Teffs from 8-week-old NOD mice. C: Similar analysis is shown of Teffs from 11-week-old mice. Depicted data represent one representative experiment done in triplicate. The error bars show the SEM. Similar results were obtained in 3–4 independent experiments. Two-tailed Student t test was used for statistical analysis: *P < 0.05, **P < 0.01, and ***P < 0.001. MFI, mean fluorescence intensity.

FIG. 3.

Reduced Ca²⁺ influx in activated NOD Teffs treated with CtxB and Gal-1. Activated cells were pretreated with N’ase and subjected to [Ca²⁺]_i measurement with fura-2 indicator. A: [Ca²⁺]_i elevation promoted by CtxB (5 μg/mL) or Gal-1 (20 μg/mL). Each curve was the average of 3–4 independent runs. B: Peak elevation of [Ca²⁺]_i, showing reduced Ca²⁺ response in NOD compared with Balb/c Teffs. The role of GM1 was demonstrated by significant recovery of Ca²⁺ response in NOD Teffs supplemented with exogenous GM1 and blockade of Ca²⁺ response in Balb/c cells pretreated with non–cross-linking anti–GM1 Ab; nonspecific IgG had no effect. Blockade of Ca²⁺ response by SK&F 96365 (100 μmol/L) indicated involvement of TRP channels, previously demonstrated to be TRPC5 (7). Data are average ± SEM (error bars) of 3–4 runs. Two-tailed Student t test was used for statistical analysis. #P < 0.05; ##P < 0.01, NOD compared with Balb/c.

FIG. 4.

Treg-induced growth inhibition of NOD Teffs mediated by Gal-1/GM1 interaction. Activated CD4⁺ and CD8⁺ Teffs were assayed for [³H]TdR uptake in the presence of variable amounts of Tregs. A: Tregs and Teffs from the same mouse showed impaired growth inhibition for NOD compared with Balb/c cells. Reactivity was restored by pretreatment of NOD Teffs with GM1. Anti–Gal-1 Ab abolished growth inhibition of Balb/c Teffs by Balb/c Tregs. B: Growth inhibition of activated Teffs by activated Tregs from the same or other strain donors. Background counts were obtained with Treg cultures only and subtracted from mixed culture counts in proportion to the number of Tregs. Responses of NOD Teffs were significantly less for both Tregs than Balb/c Teffs. Statistical analysis: one-way ANOVA with repeated measurements and the Tukey multiple comparison post-test. The error bars show the SEM.

FIG. 5.

Growth inhibition of Teffs by GM1 cross-linking agents. A: Activated CD4⁺ and CD8⁺ Teffs were assayed for [³H]TdR uptake in the presence of variable amounts of CtxB and Gal-1. Balb/c Teffs showed CtxB and Gal-1 dose-dependent inhibition of cell growth, whereas NOD Teff showed significantly less inhibition to both reagents. NOD Teff inhibition was significantly enhanced by GM1 pretreatment. Statistical analysis: one-way ANOVA with repeated measurements and the Tukey multiple comparison post-test. B: Robust response of Balb/c Teffs to CtxB or Gal-1 was blocked by non–cross-linking anti–GM1 IgG mAb, but not by nonspecific IgG. Statistical analysis: two-tailed Student t test; *P < 0.01, **P < 0.001 compared with corresponding controls. The error bars show the SEM.