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. 2007 Nov;122(3):409-17.
doi: 10.1111/j.1365-2567.2007.02655.x. Epub 2007 Jul 6.

Innate immunity modulates autoimmunity: type 1 interferon-beta treatment in multiple sclerosis promotes growth and function of regulatory invariant natural killer T cells through dendritic cell maturation

Gianluigi Gigli  1 , Simone Caielli, Daniela Cutuli, Marika Falcone
Affiliations

Innate immunity modulates autoimmunity: type 1 interferon-beta treatment in multiple sclerosis promotes growth and function of regulatory invariant natural killer T cells through dendritic cell maturation

Gianluigi Gigli et al. Immunology. 2007 Nov.

Abstract

Type 1 interferon-beta (T1IFN-beta) is an innate cytokine and the first-choice therapy for multiple sclerosis (MS). It is still unclear how T1IFN-beta, whose main function is to promote innate immunity during infections, plays a beneficial role in autoimmune disease. Here we show that T1IFN-beta promoted the expansion and function of invariant natural killer (iNKT) cells, an innate T-cell subset with strong immune regulatory properties that is able to prevent autoimmune disease in pre-clinical models of MS and type 1 diabetes. Specifically, we observed that T1IFN-beta treatment significantly increased the percentages of Valpha24(+) NKT cells in peripheral blood mononuclear cells of MS patients. Furthermore, iNKT cells of T1IFN-beta-treated individuals showed a dramatically improved secretion of cytokines (interleukins 4 and 5 and interferon-gamma) in response to antigenic stimulation compared to iNKT cells isolated from the same patients before T1IFN-beta treatment. The effect of T1IFN-beta on iNKT cells was mediated through the modulation of myeloid dendritic cells (DCs). In fact, DCs modulated in vivo or in vitro by T1IFN-beta were more efficient antigen-presenting cells for iNKT cells. Such a modulatory effect of T1IFN-beta was associated with up-regulation on DCs of key costimulatory molecules for iNKT (i.e. CD80, CD40 and CD1d). Our data identified the iNKT cell/DC pathway as a new target for the immune regulatory effect of T1IFNs in autoimmune diseases and provide a possible mechanism to explain the clinical efficacy of T1IFN-beta in MS.

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Figures

Figure 1
Figure 1
Percentage of Vα24+ CD3+ NKT cells increased in PBMC of MS patients after treatment with T1IFN-β. (a) Cumulative data of percentages of iNKT in healthy individuals and MS patients before and after treatment with T1IFN-β are presented. Total peripheral blood mononuclear cells (PBMC) were stained with PE-conjugated anti-CD3 and FITC-conjugated anti-Vα24 monoclonal antibodies and analysed by flow cytometry. Percentages of CD3+ Vα24+ iNKT cells significantly increased in T1IFN-β-treated patients (n = 8) compared to PBMC samples from untreated individuals (untreated MS n = 8 and healthy controls, n = 8) (non-parametric Mann–Whitney test, P = 0·04). (b) Percentages of Vα24+ CD3+ iNKT cells increased progressively in the PBMC of MS patients after treatment with T1IFN-β. PBMC samples of each patient (open squares) and controls (closed squares) collected at different time-points were stained and analysed in a single experiment as in (b) (c) The majority of invariant Vα24+ T cells were αGalCer/CD1d+. Total PBMC of MS patients (n = 3) and controls (n = 3) were double-stained with PE-conjugated αGalCer-loaded CD1d tetramers and FITC-conjugated anti-Vα24 mAb. The small percentage (5–15%) of Vα24+ T cells that were αGalCer/CD1d– could represent CD1d-restricted iNKT cells that did not recognize αGalCer as their glycolipid antigen.
Figure 2
Figure 2
T1IFN-β treatment in MS patients promoted iNKT cell function. The iNKT cells purified from T1IFN-β-treated MS patients (T1IFN-β MS; n = 8) secreted significantly larger amounts of IL-4 (P ≤ 0·01), IL-5 (P ≤ 0·01) and IFN-γ (P ≤ 0·05) compared to their counterparts from untreated healthy controls (n = 8) or the same MS patients before they started treatment (untreated MS; n = 8). IL-10 secretion increased slightly, but not significantly, in T1IFN-β-treated MS patients compared to untreated controls or MS patients (P ≤ 0·09). Vα24+ iNKT cells were expanded by culturing PBMC with αGalCer (100 ng/ml) and cytokines (recombinant human IL-7 and IL-15) and purified by magnetic bead separation with anti-Vα24 monoclonal antibody. iNKT cells (2 × 104 per well) were plated on a 96-well plate and stimulated with 2 × 105αGalCer-pulsed DCs. Supernatants were collected after 7 days and cytokines were simultaneously measured in the supernatants of iNKT cell cultures by a multiplexed flow cytometric assay (Cytometric Bead Array).
Figure 3
Figure 3
T1IFN-β did not directly enhance NKT cell proliferation and cytokine secretion. The addition of T1IFN-β to iNKT cell cultures inhibited both antigen-specific proliferation and cytokine secretion. Purified Vα24+ iNKT cells (2 × 104; previously expanded in vitro from PBMC of untreated individuals) were stimulated with unpulsed (– αGalCer) or antigen-pulsed (+ αGalCer), irradiated DCs (2 × 105) in 96-well plates with/without rhT1IFN-β (1000 U/ml). (a) Proliferation was measured by 3[H]thymidine incorporation in the last 16 hr of a 3-day culture. (b) Supernatants were collected after 7 days and cytokine release (IL-5) was measured by CBA assay.
Figure 4
Figure 4
T1IFN-β acted on DC by increasing their stimulatory capacity upon iNKT cells. (a) Pre-conditioning of monocytes with T1IFN-β in treated MS patients favoured differentiation of DCs with an enhanced antigen-presenting capacity upon iNKT cells compared to DCs from untreated MS patients or healthy controls (P ≤ 0·05). PBMC were collected from MS patients before (MS) and 3 months after treatment with T1IFN-β (T1IFN-β/MS) and kept for 2 hr at 37°, in 5% CO2 in RPMI-1640 with 10% fetal calf serum. Adherent cells were cultured for 5 days with rhGM-CSF (400 U/ml) and rhIL-4 (200 U/ml) to differentiate DCs. DCs were either unpulsed or were pulsed with the iNKT cell antigen (± αGalCer, 100 ng/ml), irradiated and cultured at 2 × 105 cells/well in a 96-well plate together with 2 × 104 purified Vα24+ iNKT cells previously expanded from autologous PBMC. Supernatants were collected after 7 days and iNKT cell stimulation was evaluated as the mean of cytokine release (IL-5 and IFN-γ) measured by CBA assay. Data are presented as mean ± SEM of duplicate determinations (SEM <5% are not shown). One representative experiment out of two is shown. (b) DCs differentiated in vitro in the presence of T1IFN-β were more effective antigen-presenting cells for iNKT cells compared to non-T1IFN-β-modulated DCs (P ≤ 0·01). DCs were differentiated from PBMC of untreated individuals (healthy donors) with rhGM-CSF and rhIL-4 with/without rhT1IFN-β, pulsed with αGalCer (100 ng/ml), irradiated and added to autologous purified Vα24+ iNKT cells. Unpulsed DCs were used as negative control (– αGalCer). Supernatants were collected after 7 days and iNKT cell stimulation was evaluated as mean of cytokine release (IL-5 and IFN-γ). Data are presented as mean ± SEM of duplicate determinations. One representative experiment out of three is shown.
Figure 5
Figure 5
T1IFN-β induced semi-maturation of resting DCs. (a) T1IFN-β up-regulated the expression of CD1d and maturation markers (CD80, CD40) on resting immature DCs. Myeloid DCs were derived from PBMC of healthy individuals with rhGM-CSF (400 U/ml) and rhIL-4 (200 U/ml) in the presence (open histogram) or absence (shaded histogram) of rhT1IFN-β (1000 U/ml). Expression of CD11c, maturation markers CD80 and CD40 and CD1d was determined by flow cytometric analysis. CD80, CD40 and CD1d histograms refer to gated CD11c+ cells. The graph at the right side of each histogram represents cumulative data of three separate determinations ±SEM. (b) T1IFN-β-conditioned DCs were not fully mature and did not secrete cytokines (IL-12 and IL-10). Cytokine release by immature DCs derived from PBMC without (iDC) or with rhT1IFN-β (T1IFN-β iDC) was assessed by CBA on supernatants of 5-day DC cultures. As positive controls, we used fully mature DCs (mDC) obtained by adding LPS (1 μg/ml) to the last 24 hr of culture.
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