Involvement of LC3-conjugation machinery in B-cell antigen receptor trafficking
LUCAS J. 1,2, FRENGER Q. 1,2, WADIER N. 1, DEPAUW S. 1, AUGE F. 3,6, KESSLER P. 4, LEFEBVRE O. 1, BLAGOJEVIC J. 5,6, KUHN L. 5,6, CHICHER J. 5,6, HAMMANN P. 5,6, GROS F. 1,2
1 INSERM UMR_S 1109 - Immuno-Rhumatologie Moléculaire, Centre de Recherche en Biomédecine de Strasbourg (CRBS), Strasbourg, France; 2 Faculté des sciences de la vie, Université de Strasbourg , Strasbourg, France; 3 Insectarium, Institut de Biologie Moléculaire et Cellulaire (IBMC), Strasbourg, France; 4 Imaging facility PICSTRA, INSERM UMR 38 (CRBS), Strasbourg, France; 5 Proteomic facility, IBMC, Strasbourg, France; 6 Centre National de la Recherche Scientifique (CNRS) , Strasbourg, France
Antigen presentation by B cells is central in the humoral immune response. B cells present antigens to helper T cells which stimulate their differentiation into plasma cells or memory cells. Autophagy-related (ATG) proteins participate in antigen presentation, but their precise contribution is still unclear. Previous work in the laboratory demonstrated the preponderant role of the ATG5 protein. We recently showed that ATG5 is involved in immobilized antigen processing after engagement by the B cell receptor (BCR) through promotion of polarized endocytosis. We aim now at precising how ATG5 protein and other partners participate in BCR trafficking.
We generated human B cell lines expressing HA-tagged ATG16L1. Using quantitative mass spectrometry, we identified novel ATG partners in the context of BCR crosslinking. Significant candidate proteins are associated with lysosome exocytosis and fusion with endosomes. We particularly focused on SNAP23, involved in lysosome exocytosis and LC3-associated phagocytosis (LAP). SNAP23 colocalization with BCR after crosslinking is impaired upon ATG5 or ATG16L1 depletion. We are now generating SNAP23-deficient B cell lines to validate its involvement in BCR trafficking and involvement in antigen presentation.
We also intended to define if LC3-conjugation to single membranes is involved in BCR trafficking and antigen acquisition. We therefore studied the impact of RUBCN depletion, essential LC3 associated phagocytosis/endocytosis protein. We stimulated primary B cells isolated from a Rubcn-/- mice, with microbeads-tethered antigens. Upon RUBCN depletion, super-resolution imaging show polarization defects of internalized BCR-containing vesicles. This suggests the involvement of RUBCN-dependent processes in BCR endocytosis.
Altogether we showed that ATG proteins polarize BCR trafficking and identify new partners in this process. We are now generating transgenic mouse models, with B cells expressing an antigen receptor specific for a model antigen to address the question of the in vivo impact on this pathway on the optimization of the humoral response.