Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Abstract

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.

FIGURE 1.

Effects of treating human Jurkat T cells with an inhibitor of glucosylceramide synthase (Genz-122346). A, treatment of Jurkat T cells with Genz-123346 for 3 days decreased the cell-surface levels of GM1 as measured by CTB labeling and flow cytometry. Solid line, mock-treated cells; dashed line, Genz-123346-treated cells. B, CD3 expression was not affected by Genz-123346 treatment as indicated by anti-CD3 antibody labeling followed by flow cytometry. No significant difference was detected between the Genz-122346-treated and untreated samples. Gray solid line, negative control; black solid line, mock-treated cells; dashed line, Genz-123346-treated cells.

FIGURE 2.

Analysis of GSLs in Jurkat T cells by LC-MS. Jurkat T cells treated with or without Genz-123346 for 3 days were collected, and their GSL content was analyzed by LC-MS. Results are expressed as mean ratios ± S.D. of GSLs in treated versus untreated cells.

FIGURE 3.

Treatment of Jurkat T cells with Genz-123346 inhibits TCR signaling and T cell activation. A, Jurkat T cells incubated with or without Genz-123346 for 3 days were activated for 0, 1, 5, and 15 min with anti-CD3/CD28 antibody-coupled Dynabeads. Cell lysates were analyzed by Western blotting for the total (t) amounts of Lck, Zap70, and LAT, as well as the amounts of the phosphorylated (p) proteins. GAPDH levels were measured to indicate loading variances. Slight backgrounds at 0 min in the phosphorylated protein samples might be due to the basal level of activation of Jurkat lymphoma T cells in culture. B, inhibition of the Ca²⁺ response by Genz-123346. Jurkat T cells treated or not with Genz-123346 for 3 days were activated as described for A, and the Ca²⁺ response was monitored as described under “Experimental Procedures.” C, Genz-123346 inhibited IL-2 production. Primary human T cells treated or not with Genz-123346 for 3 days were activated with Dynabeads as described for A and incubated for 24 h. IL-2 levels in the media determined by ELISA and are expressed as means ± S.D.

FIGURE 4.

Reduction of GSLs by Genz-123346 inhibits TCR signaling without affecting the integrity of lipid rafts. Genz-123346-treated or untreated Jurkat T cells were activated with Dynabeads for 0 and 5 min, lysed, and fractionated by ultracentrifugation. Equal volumes of each fraction were analyzed by Western blotting using antibodies to LAT or flotillin (a marker of lipid rafts). Flotillin in the Genz-122346-treated or untreated cells was similarly confined to fractions 2–5, mainly in fractions 3 and 4. Fraction 1 represents the top of the gradient, and fraction 12 represents the bottom. Results are representative of three separate experiments. A, untreated cells; B, Genz-123346-treated cells; C, ratios of total (t) LAT and phosphorylated (p) LAT between treated and untreated cells of a representative study.

FIGURE 5.

Reduced GSL levels in GM3S-deficient T cells attenuates T cell activation and proliferation. CD4⁺ T cells were isolated from splenocytes of GM3S wild-type littermates (+/+) and GM3S heterozygous (+/−) and KO (−/−) mice. Cells were plated onto U-bottom 96-well plates precoated with anti-CD3 antibody and supplemented with soluble anti-CD28 antibody. A, after 24 h, IL-2 levels in the media were measured by ELISA. B, the proliferation status of the cells was measured by [³H]thymidine incorporation. Results are representative of two experiments. Data are presented as means ± S.D. *, p < 0.05 between GM3S^+/+ and GM3S^−/− mice.

FIGURE 6.

Reduction of GSL levels by Genz-123346 inhibits Th₁₇ differentiation. Naive CD4⁺CD62L⁺ T cells from wild-type C57BL/6 mice were used to assess the effects of Genz-123346 on Th differentiation. A, effect of Genz-123346 on differentiation of wild-type naive T cells into Th₁ and Th₁₇ subsets. Intracellular cytokines were stained using fluorescently labeled antibodies. Plots depict the expression of IFN-γ and IL-17 in live CD4⁺ gated cell populations. B, effect of Genz-123346 on Th₁₇ differentiation as assessed by RORγt intracellular staining and IL-17 production. CTB denotes the GM1 level on the cell surface, which was reduced by Genz-123346 treatment. Results are representative of at least three studies.

FIGURE 7.

Reduction of GSL levels through inactivation of GM3S inhibits Th₁₇ differentiation. Naive CD4⁺CD62L⁺ T cells from GM3S KO (−/−) and heterozygous (+/−) mice and wild-type littermates (+/+) on a C57BL/6-129/svev mixed background were prepared and differentiated into various helper T cell subsets as described under “Experimental Procedures.” A, effect of GM3S deficiency on differentiation of naive T cells into Th₁ and Th₁₇ subsets. B, IL-17 levels in culture media of samples in A as measured by ELISA. Results are representative of at least three studies.