UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

Sophia Maschalidi,Sophia Maschalidi,Sophia Maschalidi,Sophia Maschalidi,Nicolas Cagnard,Nicolas Cagnard,Nicolas Cagnard,Peter Van Endert,Peter Van Endert,Peter Van Endert,Peter Van Endert,Peter Van Endert,Thierry Capiod,Thierry Capiod,Thierry Capiod,Thierry Capiod,Thierry Capiod,Clarissa R Nascimento,Nicolas Demaurex,Paula Nunes-Hasler,Ignacio Sallent,Ignacio Sallent,Ignacio Sallent,Ignacio Sallent,Ignacio Sallent,Bénédicte Manoury,Bénédicte Manoury,Bénédicte Manoury,Bénédicte Manoury,Bénédicte Manoury,Fernando E Sepulveda,Fernando E Sepulveda,Fernando E Sepulveda,Fernando E Sepulveda,Pablo Vargas,Pablo Vargas,Pablo Vargas,Valérie Lannoy,Valérie Lannoy,Valérie Lannoy,Valérie Lannoy,Valérie Lannoy,Ana-Maria Lennon-Duménil,Guillaume Darrasse-Jèze,Guillaume Darrasse-Jèze,Guillaume Darrasse-Jèze,Guillaume Darrasse-Jèze,Guillaume Darrasse-Jèze,Meriem Garfa-Traore,Meriem Garfa-Traore,Meriem Garfa-Traore

doi:10.1038/s41467-017-01601-5

Abstract

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

Highlights

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells
In professional antigen-presenting cells such as dendritic cells (DCs) or macrophages, exogenous antigens can be presented by MHC class I (MHCI) molecules, a process described as the cross-presentation pathway[1,2]
In WT cells, we observed a Ca2+ influx that was reduced by 50% in DCs silenced for STIM1 (Fig. 6c), to what we found in 3d/3d DCs (Fig. 4d)

Summary

60 Min OVA-coated beads OVA-coated spleen

Silencing STIM1 in DCs inhibits antigen cross-presentation. To investigate the contribution of STIM1 in cross-presentation, we knocked down the expression of STIM1 in BM-DCs using four different siRNA, with siRNA #1 and #5 being the most potent at reducing Stim[1] mRNA levels without interfering with Stim[2] mRNA (Fig. 6a). To assess antigen cross-presentation, DCs from WT and 3d/3d mice transfected with control (non-targeting) or Stim[1] siRNA were incubated with OVAb or OVA-coated splenocytes, co-cultured with CFSE-labeled OT-I T cells and T cell activation was measured. Fibroblasts lacking STIM1 were unable to cross-present OVA-pICS, while SIINFEKL peptide was presented by both control and STIM1-deficient fibroblasts (Fig. 6h). These results strongly suggest that STIM1 is required for OVA degradation and cross-presentation in murine and human cells. We conclude that interfering with antigen degradation either by restoring STIM1 activity in 3d/3d cells or by inhibiting endosomal acidification upon ConB addition in WT DCs has a major impact in antigen cross-presentation

Discussion

Findings

Methods