The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I–peptide complex formation

Abstract

CD8α+ dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α+ DCs, and Siglec-G deficient (Siglecg−/−) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I–peptide complexes were more abundant on Siglecg−/− CD8α+ DCs than on Siglecg+/+ CD8α+ DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47phox and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I–peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity.

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Figure 1: Much lower expression of Siglec-G in CD8α+ splenic DCs.
Figure 2: Enhanced CTL responses to LM infection in Siglecg−/− mice.
Figure 3: Siglecg−/− mice exhibit a more potent CD8+ T cell response to BCG infection and tumor inoculation.
Figure 4: Siglecg−/− DCs induce more-potent antigen-specific CTL proliferation via enhanced cross-presentation.
Figure 5: Siglec-G inhibits DC cross-presentation by impairing formation of the MHC class I–peptide complex.
Figure 6: Siglec-G promotes excessive antigen degradation.
Figure 7: Siglec-G recruits SHP-1 to dephosphorylate p47phox, which inhibits NOX2 activation in the phagosomes of DCs.

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Acknowledgements

We thank Y. Liu (Children's National Medical Center, Washington) for Siglecg−/− mice; H. Shen (University of Pennsylvania) for LM; H.Nie (Shanghai JiaoTong University) for the B16-OVA cell line; G. Liu (Chinese Academy of Sciences) for the plasmid pKSV7; S. Xu, Z. Jiang for technical assistance; and T. Chen and L. Lu for discussions. Supported by the National Key Basic Research Program of China (2013CB530502 and 2014CB542101), the National Natural Science Foundation of China (81522019, 31390431, 31270966 and 81471567) and the Shanghai Pujiang Program (14PJ1410800).

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Y.D., Z.G., Y.L., X.L. Q.Z., X.X., Y.G., Y.Z., and D.Z. performed the experiments; Y.D., Z.G. and X.C. analyzed data and wrote the manuscript; and X.C. designed and supervised the research.

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Correspondence to Xuetao Cao.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Siglecg−/− DCs induce antigen-specific CTL proliferation more potently.

(a,b) Splenic CD8+DCs sorted from Siglecg+/+ or Siglecg−/− mice were pulsed with either 3μm OVA protein beads or OVA(257–264) beads then co-cultured with naive OT-I T cells at a 1:1 ratio. The absolute number of live OT-I T cells (a) and IFN-γ levels in the supernatants (b) of each group were analyzed 2 days later. (c) Splenic CD8+DCs from Siglecg+/+ or Siglecg−/− mice were co-cultured with naive OT-II CD4+ T cells at a 1:1 ratio with soluble OVA protein or OVA(323-339), and the absolute number of live OT-II T cells was analyzed by flow cytometry 3 days later. *P<0.05, **P<0.01 (two tailed t-test). The data are from one experiment representative of two independent experiments with similar results (mean + s.e.m.).

Supplementary Figure 2 Siglec-G does not affect IFN-β production by splenic CD8+ DCs.

Splenic CD8+DCs sorted from Siglecg+/+ or Siglecg−/− mice were infected with LM-OVA or stimulated with LPS in vitro, and IFN-β in the supernatants was determined by ELISA 8 hours later. Data are representative of two separate experiments (mean + S.E.M).

Supplementary Figure 3 Typical standard curves for analysis of phagosomal pH in DCs.

Representative standard curves for Siglecg+/+ and Siglecg−/− CD8+ DCs for phagosomal pH calibration. Data are representative of two separate experiments.

Supplementary Figure 4 Model of the negatively regulation of DC cross-presentation by Siglec-G.

The normal phagosomal pH helps keep an environment for low protein degradation to maintain the normal pathogen proteolysis speed, then the exogenous antigenic isotope can be recognized and loaded onto MHC class I molecules, forming a MHC class I-peptide complex and the information of exogenous antigen could be cross-presented to the CD8+T cells for initiation of adaptive immune response. However, at the beginning of the infection, antigens could up-regulate the expression of Siglec-G on DCs. Siglec-G on phagosomes could use the ITIM domain to recruit SHP1 which dephosphorylates p47phox, the subset of NOX2, and consequently inhibits NOX2 activation and ROS production. So the pH of phagosomes drops and the levels of proteolysis rise, high proteolysis and low pH destroy MHC class I-restricted epitopes. As a result, Siglec-G impairs the formation of the MHC class I–peptide complex and CD8α+DC cross-presentation, finally inhibiting the initiation of adaptive immune response.

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Ding, Y., Guo, Z., Liu, Y. et al. The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I–peptide complex formation. Nat Immunol 17, 1167–1175 (2016). https://doi.org/10.1038/ni.3535

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