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Comparative Study
. 2017 Jan 24;8(4):6857-6872.
doi: 10.18632/oncotarget.14314.

N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo

Christoph Kreer  1 , Janina M Kuepper  2 , Matthias Zehner  1 , Thomas Quast  1 , Waldemar Kolanus  1 , Beatrix Schumak  2 , Sven Burgdorf  1
Affiliations
Comparative Study

N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo

Christoph Kreer et al. Oncotarget. .

Abstract

N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far.To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells.These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii.

Keywords: Komagataella phaffii; T cell activation; antigen presentation; cross-presentation; receptor-mediated endocytosis.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Glycan-dependent binding of the MR to β-gal expressed in K. phaffii
A. Binding of MR-CTLD, MR-Nterm or the Fc part of a human IgG isotype control to OVA, PNGase-treated OVA, collagen or BSA was determined by ELISA. B. OVA and PNGase F-treated OVA were separated by SDS-PAGE and subjected to coomassie staining or far-western blot analysis using 50 μg/ml MR-CTLD. C. BM-DCs were incubated with fluorochrome-labeled OVA or PNGase F-treated OVA and analyzed by flow cytometry. D. Scheme of β-gal expressed by K. phaffii. MF: Mating Factor alpha leader sequence, FLAG: FLAG-epitope. E. Purification of β-gal(V10-L731) from K. phaffii by affinity chromatography. F. Binding of MR-CTLD, MR-Nterm or isotype control to β-gal, OVA, collagen or BSA was determined by ELISA. G. as in F) using glycosylated or de-glycosylated β-gal. H. Far-western blot analysis using MR-CTLD on untreated, PNGase F-treated Endo H-treated β-gal from K. phaffii and E. coli. Bar graphs show mean values ± SEM. All graphs depict representative examples of at least 3 independent experiments.
Figure 2
Figure 2. MR-mediated uptake of β-gal expressed in K. phaffii
A. Internalization of β-gal from K. phaffii by HEK-MR or control HEK293T (HEK) cells monitored by flow cytometry. B and C. Internalization of β-gal by wildtype or MR-deficient BM-DCs monitored by flow cytometry (B) or fluorescence microscopy (C). Nuclei (blue) were visualized with 1 μg/ml 4',6-diamidino-2-phenylindole (DAPI). Bars: 20 μm. D. Uptake of β-gal by wildtype BM-DCs in the presence of 3 mg/ml mannan or 5 mM EDTA. E. Uptake of untreated or Endo H-treated β-gal by wildtype or MR-deficient BM-DCs. F. BM-DCs were incubated with fluorochrome-labeled β-gal and OVA and analyzed by fluorescence microscopy. Nuclei are depicted in blue. Bars: 10 μm. G. Co-localization of β-gal with Trf, EEA1, the MR and LAMP-1 depicted by fluorescence microscopy. Graphs show mean values ± SEM. All graphs depict representative examples of at least 3 independent experiments.
Figure 3
Figure 3. Introduction of an artificial N-glycosylation site in GFP and expression in K. phaffii results in binding and endocytosis by the MR
A. Scheme of K. phaffii -expressed GFP constructs. MF: Mating Factor alpha leader sequence. B. Binding of MR-CTLD, MR-Nterm or isotype control to NST-GFP, OVA, collagen or BSA depicted by ELISA. C. Binding of MR-CTLD to untreated or Endo H-treated NST-GFP by ELISA. D. Binding of MR-CTLD to untreated, PNGase F-treated or Endo H-treated NST-GFP or QST-GFP depicted by far-western blot analysis. E. Uptake of NST-GFP by wildtype or MR-deficient DCs in the presence or absence of 3 mg/ml mannan. F. Uptake of untreated or Endo H-treated NST-GFP by wildtype or MR-deficient BM-DCs. G. Binding of MR-CTLD to untreated or PNGase F-treated NST-GFP or QST-GFP from K. phaffii or HEK293T cells depicted by far-western blot analysis. H. Uptake of NST-GFP from K. phaffii and HEK293T cells by wildtype and MR-deficient DCs. I. Fluorescence microscopy of BM-DCs incubated with NST-GFP and fluorochrome-labeled OVA. Nuclei are depicted in blue. Bar: 10 μm. Graphs show mean values ± SEM. All graphs depict representative examples of at least 3 independent experiments.
Figure 4
Figure 4. Recognition of N-glycosylated proteins does not influence DC maturation
A. BM-DCs were treated with 100 μg/ml β-gal, 100 μg/ml Endo H-treated β-gal or 0.1 μg/ml LPS. Secretion of TNFα, IL-12 and IL-6 was determined after 18h by ELISA. B. BM-DCs were treated with 4 μg/ml untreated β-gal or Endo H-treated β-gal from K. phaffii, with 4 μg/ml β-gal from E. coli or with 1 μg/ml LPS. Expression of MHC I, CD40, CD80 and CD86 was monitored after 18 h by flow cytometry. C. BM-DCs were stimulated with untreated β-gal, Endo H-treated β-gal, LPS or medium only. After intense washing, cells were pulsed with OVA257-264 (left) or OVA323-339 (right) and incubated with OT I (left) or OT II (right) T cells. T cell activation was measured after 18 h by ELISA. D. Identical to C) using CFSE-labeled T cells. T cell proliferation was measured after 3 days by flow cytometry. Graphs show mean values ± SEM of three technical replicates. Graphs depict representative examples of at least 3 independent experiments.
Figure 5
Figure 5. Effect of N-glycosylation on antigen presentation critically depends on the nature of the antigen
A. Scheme of in vivo experiments. s.c.: subcutaneously, i.v.: intravenously. B. Mice were immunized with N-glycosylated or de-glycosylated β-gal as depicted in A). β-gal-specific cytotoxicity was determined after injection of a mixture of differentially labeled target and control cells by flow cytometry (n=28). C. Cells from draining lymph nodes from mice treated as in A) were isolated and re-stimulated with 2 μM of the β-gal epitope ICPMYARV for 18 h. Secretion of IFNγ in the supernatant was determined by ELISA (n=12). D. Mice were immunized with N-glycosylated or de-glycosylated NST-GFP-S8L as in A). Cytotoxicity was determined by flow cytometry (n=6). E and F. Mice were treated as in D) and IFNγ (E) or IL-4 (F) secretion was determined after re-stimulation with SIINFEKL (n=6). G. Binding of MR-CTLD to untreated or Endo H-treated NST-GFP-S8L and QST-GFP-S8L (purified from K. phaffii) depicted by far-western blot analysis. H. Binding of MR-CTLD to untreated or Endo H-treated 4xNST-GFP-S8L and NST-GFP-S8L depicted by far-western blot analysis. I. Mice were immunized with untreated or Endo H-treated 4xNST-GFP-S8L as in A). Secretion of IFNγ after re-stimulation with SIINFEKL was determined by ELISA (n=6 per group). J. As in H using β-gal-S8L. K. As in I) but with β-gal-S8L (n=6). L. As in H using NST-GFP-TRP2. M. As in I but after injection of NST-GFP-TRP2 and restimulation with SVYDFFVWL (n=6).
Figure 6
Figure 6. Influence of N-glycosylation on the induction of a humoral immune response
A-E. IgG against β-gal (A), NST-GFP-S8L (B), 4xNST-GFP-S8L (C), β-gal-S8L (D) or NST-GFP-TRP2 (E) from the serum of mice treated as in Figure 5A was determined by ELISA (n≥6). F and G. Mice were injected with CFSE-labeled OT II (F) or OT-I (G) and challenged with NST-GFP-I17R (F) or NST-GFP-S8L (G). T cell proliferation was monitored by flow cytometry after 3 days. H-J. Mice were immunized with untreated or Endo H-treated NST-GFP-I17R as indicated in Figure 5A. Secretion of IFNγ (H), IL-4 (I) or IL-6 (J) secretion was determined by ELISA after re-stimulation with ISQAVHAAHAEINEAGR (n=6). All graphs show mean values ± SEM.
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