Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

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1) Questions arose regarding the identity of the compartment of cross-presentation. Using kinetics experiments, the authors claim that Ova-Le^x re-routes antigen from an early EEA1⁺Rab11⁺ endosome to an LAMP1⁺Rab11⁺ endolysosomal compartment where antigen lasts for long periods of time. To formally prove that Le^x modification results in re-routing of antigen, the authors should include the routing kinetics of native Ova.

We have performed additional experiments to compare the intracellular routing of OVA-Le^X with that of OVA at different time points by confocal laser scan microscopy. Co-staining with EEA-1, LAMP-1 and Rab11 revealed that upon internalization, OVA-Le^X is rapidly shuttled to EEA-1⁺Rab11⁺ compartments from where it moves to LAMP-1⁺Rab11⁺ compartments after 2 hrs (see Figure 6—figure supplement 3A). In contrast, OVA-Le^X does not end up in LAMP-1⁺Rab11⁺ compartments in MGL1 KO BM-DCs (Figure 6—figure supplement 3C). Importantly, these data confirm our imaging flow cytometric analysis (Figure 6A and B in the revised manuscript). While OVA also rapidly routes to EEA-1⁺Rab11⁺ compartments, it does not move to LAMP-1⁺Rab11⁺ at later time points, in contrast to OVA-Le^X (Figure 6—figure supplement 3B). This indicates that the modification of OVA with Le^X that binds MGL1 alters the intra-cellular routing of the antigen.

The new confocal data obtained on the differential intracellular routing of OVA-Le^X and OVA are added to the manuscript (Results, subsection “Modification of OVA with LeX alters the intracellular routing of OVA” and Figure 6, panel C. Additionally, a more detailed representation of the confocal analysis we performed is shown in Figure 6—figure supplement 3). Furthermore, we have adapted the title of the manuscript to “Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells.”

2) As presented in Figure 3B and 3C, the results neither prove nor suggest whether cross-presentation occurs in EA1⁺Rab11⁺ endosome, the LAMP1⁺Rab11⁺ endolysosomal compartment or both. It would strengthen the manuscript substantially to provide some functional evidence for cross-presentation in these compartments.

Indeed, the reviewers are correct that the data as presented as well our new confocal imaging data do not show that OVA-Le^X is cross-presented within Rab11⁺LAMP-1⁺ compartments. We therefore weakened the Discussion as we do not want to claim that cross-presentation is occurring there. To analyze this in depth we would need to generate Rab11-deficient (WT and MGL1 KO) BM-DCs by CRISPR-Cas9 technology as well as further identification of this compartment, which we consider out of the scope of this manuscript.

3) It is particularly important that the authors demonstrate by confocal imaging the co-localization of Ovalbumin Le^x in Rab11⁺Lamp1⁺ compartments in WT and MGL-1^-/- BMDCs, as the resolution of the FACS based immuno-fluorescence analyses is less convincing.

As mentioned above, we have performed additional experiments to analyze the intracellular routing of OVA-Le^X at different time points by confocal laser scan microscopy. These new data confirm our imaging flow cytometric analysis (Figure 6A+B of the revised manuscript) as they show that upon internalization, OVA-Le^X is rapidly shuttled to EEA-1⁺Rab11⁺ compartments from where it moves to LAMP-1⁺Rab11⁺ compartments (see Figure 6—figure supplement 3A below). Moreover, neither OVA nor OVA-Le^X in MGL1 KO BM-DCs does end up in LAMP-1⁺Rab11⁺ compartments (Figure 6—figure supplement 3B+C). Thus, together these data strongly suggest that OVA modified with Le^X, and internalized via MGL1 is differently routed intra-cellular. These confocal images are added to the manuscript (Results, subsection “Modification of OVA with LeX alters the intracellular routing of OVA” and Figure 6, panel C. Additionally, a more detailed representation of the confocal analysis is shown in Figure 6—figure supplement 3).

4) It is also critical that the authors compare MGL-1-Fc with a Mannose receptor Fc fusion protein and recognition of Ova.

We have performed these additional experiments, and indeed confirm that the MR equally binds to OVA and OVA-Le^X, while MGL1-Fc specifically binds to OVA-Le^X (see Author response image 1). These data further strengthen the previous binding of the MR to endogenous glycans on OVA (Burgdorf et al., 2007. Science, 316, 612-616) and show that modification of OVA with Le^X increases its affinity to bind MGL1, favoring efficient cross-presentation, independent of MyD88 and Cathepsin-S.

The comparison of MR-Fc with MGL-1-Fc binding to OVA-Le^X and OVA has been added to the manuscript (Results, subsection “Identification of glycans on native and glycan-modified OVA, and the consequences for CLR-specific binding and cross-presentation” and Figure 1, panel B).

Author response image 1

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5) Apart from performing these additional experiments in support of the study claims, the authors need to re-analyze several results, e.g., Figures 2B,C,D, 3, 4, etc., and in other instances show representative flow cytometry data.

We have re-analyzed the data shown in Figures 2, 3 and 4 of the manuscript. Based on this re-analysis only the scatter plot showing the frequency of IFN-γ-producing CD8⁺ T cells has been adapted (Figure 2C). Representative pictures of flow cytometric analysis as well as adapted scatter plot have been added to the manuscript (Figure 2—figure supplements 1 and 2, Figure 3—figure supplement 2, Figure 4—figure supplements 1 and 3).