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, 155 (3), 379-386

Interleukin-6 Signalling Mediates Galectin-8 Co-Stimulatory Activity of Antigen-Specific CD4 T-cell Response

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Interleukin-6 Signalling Mediates Galectin-8 Co-Stimulatory Activity of Antigen-Specific CD4 T-cell Response

Julieta Carabelli et al. Immunology.

Abstract

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect.

Keywords: T-cell receptor co-stimulation; antigen-presenting cells; galectins; phosphorylated signal transducer and activator of transcription 3.

Figures

Figure 1
Figure 1
Galectin‐8 (Gal‐8) induces interleukin‐6 (IL‐6) secretion during co‐stimulation of antigen‐specific CD4 T‐cell response. (a) Quantification of IL‐6 by ELISA in supernatants from Gal‐8‐induced co‐stimulation cultures. (b) Quantification of interferon‐γ (IFNγ) by ELISA in supernatants (left) and cell proliferation (right) of Gal‐8‐induced co‐stimulation cultures. For all assays, splenocytes (3 × 105 cells) from DO11.10 mice were cultured for 48 hr in the presence of 0·1 μg/ml of ovalbumin323–339 peptide (OVA), and/or 0·2 μm of Gal‐8. Thiodigalactoside (TDG, 30 mm) was added 30 min before the stimulus. TDG had no effect on OVA response (not shown). ND, not detected. Depicted assays are representative of at least three independent experiments and were carried out, each time, with different recombinant protein preparations. **P < 0·01; ***P < 0·001; ****P < 0·0001.
Figure 2
Figure 2
Antigen‐presenting cells (APC) produce interleukin‐6 (IL‐6) during galectin‐8 (Gal‐8) ‐induced co‐stimulation. IL‐6 intracellular expression was determined by flow cytometry during Gal‐8‐induced co‐stimulation. Splenocytes (4 × 106) from DO11.10 mice were cultured for 24 hr in the presence of 0·1 μg/ml of ovalbumin323–339 (OVA 323–339) peptide and 0·2 μm of Gal‐8 (OVA + Gal‐8), or left untreated (Control). Cells were labelled with specific monoclonal antibodies for surface antigens (MHCII, CD11b, CD11c, F4/80, CD4 and B220) and for intracellular IL‐6. IL‐6+ population is box enfolded in the counter plots. Bars indicate percentage of IL‐6+ cells and the mean fluorescence intensity (MFI). FSC, forward side scatter. Depicted assays are representative of two independent experiments and were carried out, each time, with different recombinant protein preparations. *P < 0·05; **P < 0·01; ***P < 0·001.
Figure 3
Figure 3
Galectin‐8 (Gal‐8) ‐induced CD4 T‐cell co‐stimulation is dependent on interleukin‐6 (IL‐6). (a) IL‐6 neutralization. Splenocytes (3 × 105 cells) from DO11.10 mice were cultured in the presence of 0·1 μg/ml of ovalbumin323–339 peptide (OVA), and/or 0·2 μm of Gal‐8, in the presence or absence of IL‐6 neutralizing monoclonal antibody (anti‐IL‐6) or isotype control (Iso). After 48 hr, proliferation was assessed. (b) Signal transducer and activator of transcription 3 (STAT3) activation. Splenocytes (9 × 106 cells) from DO11.10 mice were stimulated for 2 or 7 hr with 0·1 μg/ml of OVA and/or 0·2 μm of Gal‐8, in the presence or absence of IL‐6 neutralizing monoclonal antibody (anti‐IL‐6) or isotype control (Iso). For positive control, cells were incubated for 15 min with 100 ng/ml of recombinant IL‐6 (rIL‐6). Protein expression levels of pSTAT3 and total STAT3 were detected by Western blot analysis. Bars depict pSTAT/STAT3 fluorescence signal ratio. (c) Gal‐8‐induced co‐stimulation in the presence of IL‐6‐deficient antigen‐presenting cells (APC). IL‐6KO and C57BL/6J mice‐derived APC (2 × 105): Mitomycin APC (upper panels) or CD4‐depleted APC (lower panels) were co‐cultured with CD4 T cells (1 × 105) purified from OTII mice, in the presence of 0·05–0·1 μg/ml of OVA and/or 0·2 μm of Gal‐8. After 48 hr, cell proliferation (right) and quantification of interferon‐γ (IFNγ) by ELISA in supernatants (left) were assessed. (d) Same as (c) with Mitomycin APC, and with addition of 5 ng/ml of rIL‐6 to the co‐cultures containing IL‐6KO APC. Fold increase was calculated as the counts/min (OVA+Gal‐8)/counts/min (OVA) ratio. Depicted assays are representative of three (a) and two (b–d) independent experiments and were carried out, each time, with different recombinant protein preparations. *P < 0·05; ***P < 0·001.

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