Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar;325:33-40.
doi: 10.1016/j.cellimm.2018.01.014. Epub 2018 Feb 2.

IFN-γ and Tumor Gangliosides: Implications for the Tumor Microenvironment

Affiliations
Free PMC article

IFN-γ and Tumor Gangliosides: Implications for the Tumor Microenvironment

Barbara Dillinger et al. Cell Immunol. .
Free PMC article

Abstract

Gangliosides shed by tumors into their microenvironment (TME) are immunoinhibitory. Interferon-γ (IFN-γ) may boost antitumor immune responses. Thus we wondered whether IFN-γ would counteract tumor ganglioside-mediated immune suppression. To test this hypothesis, we exposed human monocyte-derived LPS-activated dendritic cells (DC) to IFN-γ and to a highly purified ganglioside, GD1a. DC ganglioside exposure decreased TLR-dependent p38 signaling, explaining the previously observed ganglioside-induced down-modulation of pro-inflammatory surface markers and cytokines. Strikingly, while increasing LPS-dependent DC responses, IFN-γ unexpectedly did not counteract the inhibitory effects of GD1a. Rather, induction of indoleamine 2,3-dioxygenase (IDO1), and expression of STAT1/IRF-1 and programmed cell death ligand (PD-L1), indicated that the immunoinhibitory, not an immune stimulatory, IFN-γ-signaling axis, was active. The combination, IFN-γ and DC ganglioside enrichment, markedly impaired DC stimulatory potential of CD8+ T-cells. We suggest that gangliosides and IFN-γ may act in concert as immunosuppressive mediators in the TME, possibly promoting tumor progression.

Keywords: Ganglioside; IFN-γ; Tumor immunosuppression; Tumor microenvironment.

Conflict of interest statement

Conflict of interest

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. GD1a ganglioside exposure inhibits co-stimulatory CD80 expression of human monocyte-derived DCs
(A) In a representative FACS histogram analysis, the median peak channel (mpc) of CD80 expression on human monocyte-derived iDCs (dashed line, mpc of 179), DCs activated with 100 ng/mL LPS and 100 U/mL IFN-γ (grey solid line, mpc 1272), and DCs exposed to 100 µM GD1a and then activated with LPS and IFN-γ (black solid line, mpc 308) are shown. (B–D) iDCs were cultured in medium alone (white bar), stimulated with LPS (100 ng/mL, dashed white bar), IFN-γ (100 U/mL, light grey bar) or with both LPS + IFN-γ (dashed grey bars) or in addition treated with GD1a (50 or 100 µM, dashed dark-grey bars). This same shading of bars has been used consistently in figures 1–4. (B), CD80; (C), CD86; (D), MHC-II expression of DCs, analyzed by flow cytometry. Bars indicate mean±SEM mpc of triplicate cultures in one representative of three separate experiments. *, P<0.05; ** P<0.01; ***, P<0.005; no significant differences in (C and D) (P>0.05) analyzed using unpaired two-tailed Student’s t test.
Figure 2
Figure 2. DC exposure to GD1a abrogates LPS-stimulated pro-inflammatory cytokine secretion and inhibits allogeneic T-cell responses, not reversed by IFN-γ
(A) DCs were stimulated as in Figure 1. Cell culture supernatants were analyzed for the cumulative release of IL-12p70, TNF-α, and IL-6. Bars indicate mean±SEM of triplicate cultures in one representative of three similar experiments. (B) DCs cultured and illustrated as in Figure 1, were washed, and then used to stimulate CFSE stained allogeneic T-cells at a DC:T-cell ratio of 1:10. The allogeneic proliferative response of live CFSE-stained CD3+CD8+ lymphocytes was assessed after 7 days of co-culture. The percent CFSE-negative cells and the mpc of CD25 were quantified by flow cytometry as exemplified in a representative experiment shown in Fig. 1B, top panel. Bars indicate mean±SEM combining four cultures of two separate experiments. Proliferation controls: resting T-cells, 0.36% CFSE negative; T-cells stimulated with anti-CD3/anti-CD28 antibodies, 93.6% CFSE negative (Data not shown). *, P<0.05; **, P<0.01, ***, P<0.005 analyzed using a two-tailed Student’s t test.
Figure 3
Figure 3. PD-L1 expression and IDO1 protein induction and activity by IFN-γ is resistant to GD1a pre-exposure of DCs
(A) DCs were cultured as in Fig. 1. At the end of culture, PD-L1 (top panel) and IDO1 (lower panel) protein expression was examined by Western blot and quantified by ImageJ; bars indicate relative PD-L1 or IDO1 expression, shown as the PD-L1/GAPDH or IDO/GAPDH ratio. One representative of two separate experiments with similar results is shown. (B) Cell culture supernatants of DCs cultured as in (A) were analyzed for tryptophan and kynurenine concentration to determine IDO1 activity. Increasing IDO1 activity is seen as reduction in tryptophan and increase in kynurenine concentration. Bars indicate the mean ±SEM of triplicate cultures in a representative of three separate experiments. ***, P<0.0005, n.s. not significant analyzed using unpaired two-tailed Student’s t test.
Figure 4
Figure 4. GD1a interferes with LPS– but not IFN-γ–initiated signaling
DCs were cultured as in Fig. 1. At the end of culture, (A) pp38, p38 (B) pSTAT1, STAT1, IRF-1 and GAPDH protein expression was determined by Western blot and quantified by ImageJ; bars indicate pp38/p38, pSTAT1/STAT1 and IRF-1/GAPDH relative protein expression.
Figure 5
Figure 5. Complex interaction of TLR-ligation, ganglioside enrichment, and IFN-γ on dendritic cell signaling
LPS/TLR-4 mediated activation of p38 drives the secretion of pro-inflammatory factors such as IL-12, TNF-α, IL-6 but also IFN-γ, through active AP1. This also involves the translocation of NFκB transcription factors into the nucleus promoting cytokine production. IFN-γ, by inducing JAK/STAT mediated signal transduction triggers the expression of IRFs, which boost cytokine production in combination with AP1 and NFκB transcription factors. IRF-1 itself is not capable of inducing cytokine production as we have also shown here. IFN-γ as well triggers transcription of IDO1 and PD-L1, inducing an immunosuppressive DC phenotype. In a ganglioside-rich environment, GD1a accumulation in DCs specifically inhibits TLR-4 signaling, strongly reducing p38-mediated AP1 activity and NFκB activity, leading to down modulation of pro-inflammatory cytokine secretion. Of note, IFN-γ signaling is not affected by GD1a enrichment, leaving JAK/STAT-mediated PD-L1 and IDO1 induction by IFN-γ intact. GD1a enriched environments therefore strongly favor immune suppressive properties of DCs in the presence of IFN-γ, which predominantly triggers the expression of enzymatically active IDO1 and PD-L1. In the figure, the hatched area indicates selective inhibitory effects of GD1a on TLR-4 signaling; the clear area depicts the unaffected IFN-γ signaling. TRAF:TNF receptor associated factor, TAK1: Transforming growth factor beta-activated kinase 1, AP1: Activator protein 1, NFκB: Nuclear factor kappa-light-chain-enhancer of activated B cells, IRF: Interferon regulatory factors.

Similar articles

See all similar articles

Cited by 2 articles

Publication types

MeSH terms

Feedback